In vitro and in vivo assay for agents which treat mucus hypersecretion

ABSTRACT

Hypersecretion of mucus in the lungs is inhibited by the administration of an epidermal growth factor receptor (EGF-R) antagonist. The EGF-R antagonist may be in the form of a small organic molecule, an antibody, or portion of an antibody that binds to and blocks the EGF receptor. The EGF-R antagonist is preferably administered by injection in an amount sufficient to inhibit formation of goblet cells in pulmonary airways. The degranulation of goblet cells that results in airway mucus production is thereby inhibited. Assays for screening candidate agents that inhibit goblet cell proliferation are also provided.

This applications claims benefit of U.S. Provisional application No.60/097,023, filed Aug. 18, 1998.

GOVERNMENT RIGHTS

The United States Government may have certain rights in this applicationpursuant to Grant HL-24136 awarded by the National Institutes of HealthProgram.

FIELD OF THE INVENTION

This invention relates generally to the field of pulmonary treatment.More particularly, the invention relates to inhibiting hypersecretion ofmucus in lungs and airways by the administration of an EGF-R antagonist.In addition, this invention also relates to methods for the developmentor assessment of candidate agents capable of inhibiting hypersecretionof mucus in the lungs.

BACKGROUND OF THE INVENTION

In the conducting airways of the respiratory system, the mucociliarysystem serves as the primary defense mechanism to move inhaled particlesor infectious agents out of the airways in the lungs. In addition,substances present in airway fluids serve to limit the toxicity of theparticles and to inactivate infective agents. The physical mechanism ofcoughing serves to expel the mucus from the airway passages (see e.g.,“Foundations of Respiratory Care,” Pierson and Kacmarek, eds. (1992)Churchill Livingstone Inc. New York, N.Y.; “Harrison's Principles ofInternal Medicine”, Fauci et al., eds. (1997) 14th Edition, McGraw Hill,New York, N.Y.

The mucociliary system consists of ciliated epithelial cells, epithelialgoblet cells, and serous and mucous cells located in submucosal glands.The cilia are surrounded by an aqueous layer (periciliary fluid)secreted into the lumen of the airway passage by the active transport ofchloride and the passive movement of water across the epithelium. Thecilia make contact with the mucus floating on this aqueous layer, andvia a unidirectional propelling motion provide for movement of mucustoward the glottis (see Pierson and Kacmarek, supra and Fauci, et al,supra). Mucus is produced by the epithelial goblet cells and submucosalgland cells and is secreted into the lumen of the airway afterdegranulation.

While mucus generally facilitates the clearance of inhaled particles orinfectious agents, hypersecretion of mucus in the airways may causeprogressive airway obstruction. In peripheral airways, cough isineffective for clearing secretions. Furthermore, because of their smalldimensions, small airways containing many goblet cells are especiallyvulnerable to airway plugging by mucus. Airway hypersecretion affects asubstantial number of individuals; it is seen in a variety of pulmonarydiseases, such as chronic bronchitis, acute asthma, cystic fibrosis, andbronchiectasis.

Hypersecretion of mucus is the major symptom in patients with chronicobstructive pulmonary disease (COPD) and defines the condition (i.e.chronic cough and sputum production). This condition alone affects 14million Americans and can cause progressive disability and death. It hasbeen estimated that asthma affects at least 4% of the U.S. populationand accounts for at least 2000 deaths annually (Pierson and Kucmarek,supra). During an acute asthmatic event, the bronchial walls swell,mucus volume increases and bronchial smooth muscle contracts, resultingin airway narrowing. As a result of hypersecretion in acute asthma,extensive mucus plugging can be a major cause of morbidity andmortality.

Hypersecretion has also been implicated in cystic fibrosis, which is oneof the most common, fatal, genetic diseases in the world. Cysticfibrosis is an autosomal recessive disease that causes the airwaymucosal cell to become unresponsive to cyclic-AMP-dependent proteinkinase activation of the membrane chloride ion channels (Pierson andKacmarek, supra and Fauci, et al., supra). The subsequent electrolyteimbalance reduces the level of hydration of the airway mucus, thusresulting in highly viscous mucus in the lungs of an individualafflicted with cystic fibrosis. Hypersecretion obstructs the airpassages of individuals with cystic fibrosis, further compromising lungfunction.

Other disease involving hypersecretion include chronic obstructive lungdisorder (COPD). Oxidant stress plays an important role in thepathogenesis of COPD. Cigarette smoke, which generates oxygen freeradicals, is strongly implicated in the pathogenesis. Neutrophils areoften seen at site of inflammation in COPD, and interestingly, oxygenfree radicals are known to be released by neutrophils during activation.

Mechanical intubation is often necessary in order to provide assistedventilation to patients with various pulmonary diseases. A tube isintroduced via the oropharanx and placed in the trachea. To preventleaking of air around the endotracheal tube, a balloon is inflatedaround the tube in the lower trachea, which may abrade the epitheliumand cause goblet cell metaplasia. Wounding of epithelium leads to repairprocesses, which can result in abundant mucus secretion. Such prolongedtracheal intubation in patients can lead to deleterious effects due tohypersecretion.

As a result of the high levels of mucus in the lungs of patients withhypersecretory pulmonary diseases, mucosal clearance is reduced.Pathological agents such as bacteria, e.g. Pseudomonas aeruginosa, oftenestablish colonies within the mucus, resulting in frequent lunginfection.

Classical modalities of treating individuals afflicted with airwayhypersecretion include antibiotic therapy, bronchodilators (e.g.,methylxanthines, sympathomimetics with strong β2 adrenergic stimulatingproperties, anticholinergics), use of systemic or inhaledcorticosteroids, primarily in asthma, liquefaction of the mucus by oraladministration of expectorants, e.g. guaifenesin, and aerosol deliveryof “mucolytic” agents, e.g. water, hypertonic saline solution (seeHarrison's, supra). A newer therapy for cystic fibrosis is theadministration of DNAse to target the DNA-rich mucus or sputum (Shak, etal. (1990) Proc. Natl. Acad. (USA) 87:9188-9192; Hubbard, R.C. et al(1991) N. Engl. J. Med. 326:812). In addition, chest physical therapyconsisting of percussion, vibration and drainage are also used tofacilitate clearance of viscous mucus. Lung transplantation may be afinal option for those with severe pulmonary impairment. Therefore, moreefficacious or alternative therapy to target the mucosal secretions isneeded. Specifically, there is a need for a specific modality that willreduce the formation of mucus secretions in the airways.

Relevant Literature

The use of EGF inhibitors to block the growth of cancer cells isreviewed by Levitski (1994) Eur J Biochem. 226(1): 1-13; Powis (1994)Pharmac. Ther. 62:57-95; Kondapaka and Reddy (1996) Mol. Cell Endocrin.117:53-58.

SUMMARY OF THE INVENTION

Hypersecretion of mucus in airways is an adverse symptom of a number ofdifferent pulmonary diseases. The secretion results from thedegranulation of goblet cells, the proliferation of which is promoted bystimulation of epidermal growth factor receptors (EGF-R). The presentinvention treats pulmonary hypersecretion by administering therapeuticamounts of EGF antagonists, preferably kinase inhibitors. Theantagonists may be in the form of small molecules, antibodies, orportions of antibodies that bind to either EGF or its receptor. Inanother aspect of the invention, in vitro and in vivo methods predictiveof the therapeutic potential of candidate agents to inhibithypersecretion of mucus are provided.

A primary object of the invention is to provide a method of treatingdiseases involving hypersecretion of mucus in lungs.

Another object of the invention is to provide formulations useful in thetreatment of diseases that result in hypersecretion of mucus.

Yet another object of the invention is to provide an in vitro assay forthe screening of candidate agents that inhibit hypersecretion of mucus,where the method involves the steps of (i) contacting an in vitro modelof goblet cell proliferation with EGF or the functional equivalentthereof; (ii) subsequently contacting the in vitro model with acandidate agent; and (iii) assessing goblet cell proliferation, whereininhibition of goblet cell proliferation is indicative of the candidateagent's therapeutic potential.

Another object of the invention is to provide an in vivo assay for thescreening of candidate agents that inhibit hypersecretion of mucus,where the method involves (i) creating an animal model of hypersecretorypulmonary disease by inducing EGF-R, e.g. with tumor necrosisfactor-alpha (TNF-α); (ii) stimulating the induced EGF-R with itsligand, e.g. transforming growth factor alpha (TGF-α) or EGF, to producemucin producing goblet cells; (iii) treating with a candidate agent; and(iv) assessing goblet cell proliferation or mucus secretion, wherein aninhibition of goblet cell proliferation or mucus secretion is indicativeof the candidate agent's therapeutic potential.

A further object of the invention is to provide in vitro and in vivoassays for the screening of EGF-R antagonists that inhibithypersecretion of mucus.

An advantage of the invention is that it provides a means for preventingexcessive formation of mucus in pulmonary airways.

A feature of the invention is that a range of different types ofantagonists can be used to block the effects of EGF and/or TGF-α andtheir interaction with EGF-R.

An aspect of the invention is formulations of EGF antagonists forreducing formation of mucus secretion in the airways of a mammalianpatient, preferably a human patient.

Another object of the invention is a method of pulmonary delivery of EGFantagonists for reducing mucus secretions in the airways of a mammalianpatient, preferably a human patient.

Another object of the invention is to provide a method for treating arange of different diseases which have as a symptom the excessformulation of mucus secretions in the airways. These diseases include,without limitation, chronic bronchitis, acute asthma, cystic fibrosis,bronchiectasis, chronic obstructive lung disease, hypersecretionresulting from epithelial damage such as allergic stimuli or mechanicalabrasions, and nasal hypersecretion.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the treatment methods, and in vitro and in vivo assaymethods, as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a western blot of EGF-R in NCI-H292 and in A431 cells. FIG.1B, immunocytochemical analysis with anti-EGF-R antibody in cultures ofNCI-H292 cells. FIG. 1C, Northern analysis of EGF-R in NCI-H292 cells.

FIG. 2. Alcian blue/PAS staining of NCI-H292 cells for identification ofmucin glycoproteins.

FIG. 3. Northern analysis for MUC5 gene expression in NCI-H292 cells.

FIG. 4A and 4B. Immunohistochemical analysis of EGF-R with an anti-EGF-Rantibody in pathogen-free rats. FIG. 4A, TNFα-treated rats. FIG. 4B,ovalbumin-sensitized rats.

FIG. 5 is a graph depicting the effect of EGF-R tyrosine kinaseinhibitor (BIBX1522) on production of goblet cells (expressed as % ofstained area of airway epithelium occupied by Alcian blue/PAS-positivestained cells).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Compositions and methods are provided for the treatment of airway mucushypersecretion by administering therapeutic amounts of EGF antagonists,preferably kinase inhibitors. The antagonists may be in the form ofsmall molecules, antibodies, or portions of antibodies that bind toeither EGF or its receptor. In airway hypersecretory diseases, e.g.chronic bronchitis, bronchiectasis, cystic fibrosis, acute asthma, COPD,etc., mucin synthesis in airways is increased and mucus hypersecretionoccurs. The secreted mucus results in airway obstruction, an effect thatcauses death in these diseases.

Several causes of airway damage and inflammation are shown herein toinduce the expression of the epidermal growth factor receptor in airwayepithelial cells. After induction of EGF-R, subsequent stimulation ofEGF-R by both ligand-dependent and -independent mechanisms results inmucin production at both gene expression and protein levels. Selectiveinhibitors of EGF-R tyrosine kinase are demonstrated to block this mucingene and protein expression.

Without limiting the invention, it is suggested that an evolutionarysequence of goblet cell production may be based on the expression ofEGF-R. Stimulation with TNFα induces intense EGF-R staining ofnon-granulated secretory cells; their subsequent activation by EGF-Rligands causes progressive staining for mucous glycoconjugates in thecytoplasm, and the cells become “pre-goblet” and then “goblet” cells.The data suggest that EGF-R activation promotes selective ceildifferentiation, but not proliferation. Goblet cells are apparentlyderived from non-granulated secretory cells that express EGF-R and arestimulated by EGF-R ligands to produce mucins.

In addition to stimulation by cytokines, the EGF-R may be stimulated byother signaling mediators. For example, prolonged cigarette smoking isassociated with progressive pathologic changes in peripheral airways,including goblet cell hyperplasia. Proinflammatory cytokine-activatedneutrophils and cigarette smoke are shown to cause mucin synthesis inhuman bronchial epithelial cells via ligand-independent activation ofEGF-R, implicating recruited neutrophils and cigarette smoke asregulators of epithelial cell differentiation that result in abnormalinduction of mucin-producing cells in airways. Neutrophils activated bya variety of stimuli, including IL-8,N-formyl-methionyl-leucyl-phenylalanine,TNF-α, cigarette smoke or H₂O₂upregulate mucin expression in epithelial cells, which synthesis isinhibited by EGF-R inhibitors. Neutrophils are also capable of producingthe EGF-R ligands, EGF and TGFα. In addition, epithelial cells aresources of EGF-R ligands.

Mechanical injury to airway epithelium can also cause hypersecretion,and be responsible for mucous plugging. Inhibitors of EGF-R tyrosinekinase serve to prevent mucous hypersecretion after tracheal intubation.

Epithelial damage is a common finding in studies of patients even withmild asthma, and the damage is increasingly related to worsening ofclinical symptoms. Epithelial damage produced by the allergic responseinduces EGF-R activation, which results in abnormal goblet cellproduction. EGF-R is implicated in epithelial damage, for example the“airway remodeling” that occurs in asthma, repair and wound closure.Mechanical epithelial damage and epithelial injury in asthma may involvea similar EGF-R cascade, resulting in abnormal growth of epithelialsecretory cells.

Hypersecretion is also an important manifestation of inflammatorydiseases of the nose. When nasal goblet cells are “challenged” byinducing goblet cell degranulation utilizing a neutrophil-dependentmechanism, expression of EGF-R and mucins are strongly upregulated.These events were associated with regranulation of the goblet cells.When inflammation, such as stimulation of neutrophil infiltration,causes goblet cell degranulation and mucin secretion, up-regulation andactivation of EGF-R re-supplies the airway epithelium with mucins.

Before the present methods of treatment and formulations are described,it is to be understood that this invention is not limited to particularmethods and formulations described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with the publications are cited.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

By “epidermal growth factor” or “EGF” is meant a protein or portionthereof having biological activity characterized by mitogenic activityon epithelial cells (e.g., Cohen (1986) Biosciences Reports 6(12):1017;Aaronson, S. A., “Growth Factors and Cancer,” Science (1991)254:1146-1153). Exemplary is the human epidermal growth factor, forexample as described by Urdea et al. (1983) Proc. Nat. Acad. Sci.80:7461-7465.

Of particular interest for the purposes of this invention is themitogenic activity of EGF on goblet cells. Also intended to beencompassed by this definition are proteins of portions thereof whichare the functional equivalent of EGF in terms of the biological responseelicited by EGF.

By “epidermal growth factor receptor” or “EGF-R” is meant a protein aportion thereof capable of binding EGF protein or a portion thereof.Exemplary is the human epidermal growth factor receptor (see Ullrich etal. (1984) Nature 309:418-425; Genbank accession number NM_(—)005228).Preferably, the binding of the EGF ligand activates the EGF-R (e.g.resulting in activation of intracellular mitogenic signaling,autophosphorylation of EGF-R). One of skill in the art will appreciatethat other ligands, in addition to EGF, may bind to EGF-R and activatethe EGF-R. Examples of such ligands include, but are not limited to,TGF-α, betacellulin, amphiregulin, heparin-binding EGF (HB-EGF) andneuregulin (also known as hergulin) (Strawn and Shawver (1998)Exp.-Opin. Invest. Drugs 7(4)553-573, and “The Protein Kinase FactsBook: Protein Tyrosine Kinases” (1995) Hardie, et al. (eds.), AcademicPress, New York, N.Y.).

By “EGF-R antagonist” is meant any agent capable of directly orindirectly inhibiting the effect of EGF-R, particularly the effect ofEGF-R on goblet cell proliferation or hypersecretion of mucus by gobletcells. EGF-R can be activated through ligand-dependent andligand-independent mechanisms, resulting in either autophosphorylationor trans-phosphorylation, respectively. EGF-R antagonists of interestmay inhibit either or both of these mechanisms. For example, binding ofTNF-α to the EGF-R results in a ligand-dependent phosphorylation, whichmay be blocked by an antibody that binds EGF-R, thereby preventing theinteraction of EGF with a ligand that would activate the EGF receptor.Examples of such antibodies are described by Goldstein et al. (1995)Clin. Cancer Res. 1:1311-1318; Lorimer et al. (1995) Clin. Cancer Res.1:859-864; Schmidt and Wels (1996) Br. J. Cancer 74:853-862. Smallmolecule tyrosine kinase inhibitors are also effective as EGF-Rantagonists.

Altematively, it is shown that compounds such as oxygen free radicalsstimulate a trans-phosphorylation of the EGF-R, resulting inligand-independent activation of the receptor. Other means of activatingEGF-R by transphosphorylation include ultraviolet and osmotic stress,stimulation of G-proten coupled-receptor by endothelin-1,lysophosphatidic acid and thrombin, m1 muscarinic acetylcholinereceptor, and human growth hormone. Antagonists of thisligand-independent mechanism include anti-oxidants, such as super oxidedismutase, DMSO, DMTU, ascorbic acid, and the like.

An EGF-R antagonist may be an antibody that binds to a factor thatstimulates EGF production or EGF-R production, thereby inhibitingpromotion of goblet cell proliferation by EGF (i.e. an inhibitor of thephosphorylation cascade that phosphorylates EGF-R). For example, afusion protein of TGFα-Pseudomonas exotoxin 40 is described by Arteagaet al. (1995) Cancer Res. 54:4703-4709.

In a preferred embodiment, the EGF-R antagonist is an inhibitor of thetyrosine kinase activity of EGF-R, particularly small moleculeinhibitors having selective action on EGF-R as compared to othertyrosine kinases—preferred small molecules block the natural EGFreceptor in a mammal, preferably a human and have a molecular weight ofless than 1 kD.

Inhibitors of EGF and EGF-R include, but are not limited to, tyrosinekinase inhibitors such as quinazolines, such as PD 153035,4-(3-chloroanilino) quinazoline, or CP-358,774, pyridopyrimidines,pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261and CGP 62706, and pyrazolopyrimidines(Shawn and Shawver, supra.),4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines (Traxler et aL, (1996) J.Med. Chem 39:2285-2292), curcumin (diferuloyl methane) (Laxmin arayana,et al, (1995), Carcinogen 16:1741-1745), 4,5-bis(4-fluoroanilino)phthalimide (Buchdunger et al. (1995) Clin. Cancer Res.1:813-821; Dinney et al (1997) Clin. Cancer Res. 3:161-168); tyrphostinscontaining nitrothiophene moieties (Brunton et al. (1996) Anti CancerDruc Design 11:265-295); the protein kinase inhibitor ZD-1839(AstraZeneca); CP-358774 (Pfizer, inc.); PD-0183805 (Warner-Lambert); oras described in International patent application WO99/09016 (AmericanCyanamid); WO98/43960 (American Cyanamid); WO97/38983 (Warener Labert);WO99/06378 (Warner Lambert); WO99/06396 (Warner Lambert); WO96/30347(Pfizer, Inc.); WO96/33978 (Zeneca); WO96/33977 (Zeneca); andWO96/33980) Zeneca; all herein incorporated by reference; or antisensemolecules.

By “inhibiting” is meant decreasing, neutralizing, attenuating orpreventing the proliferation of goblet cells, degranulation of gobletcells or hypersecretion of mucus by goblet cells.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylacticin terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes:

(a) preventing the disease or symptom from occurring in a subject whomay be predisposed to the disease or symptom but has not yet beendiagnosed as having it;

(b) inhibiting the disease or symptom, i.e., arresting its development;or

(c) relieving the disease or symptom, i.e., causing regression of thedisease or symptom. The invention is directed toward treating patientswith pulmonary or airway disease and is particularly directed towardtreating patients' hypersecretion of mucus, i.e. preventing, inhibitingor relieving hypersecretion of mucus. In terms of treating symptoms, theinvention is directed toward decreasing mucus or sputum in the airways,inhibiting infection by pathological organisms, alleviating cough, andpreventing hypoxia due to airway plugging.

More specifically “treatment” is intended to mean providing atherapeutically detectable and beneficial effect on a patient sufferingfrom a pulmonary disease involving hypersecretion of mucus.

Still more specifically “treatment” shall mean preventing, alleviating,and/or inhibiting hypersecretion of mucus with a compound selected fromthe group consisting of EGF and/or EGF-R antagonists such as antibodies,protein tyrosine kinase inhibitors and antisense molecules and the like.An alternative treatment may comprise prevention of EGF-R expression inairway, thereby blocking the pathway at an earlier stage. For example,reagents that block binding of TNFα to its receptor may preventupregulation of EGF-R.

Treatment includes preventing or inhibiting infections by pathologicalagents caused by and/or related to hypersecretion of mucus.

By “antibody” is meant an immunoglobulin protein that is capable ofbinding an antigen. Antibody as used herein is meant to include antibodyfragments, e.g. F(ab′)2, Fab′, Fab, capable of binding the antigen orantigenic fragment of interest. Preferably, the binding of the antibodyto the antigen inhibits the activity of EGF or EGF-R.

The term “humanized antibody” is used herein to describe completeantibody molecules, i.e. composed of two complete light chains and twocomplete heavy chains, as well as antibodies consisting only of antibodyfragments, e.g. Fab, Fab′, F (ab′) 2, and Fv, wherein the CDRs arederived from a non-human source and the remaining portion of the Igmolecule or fragment thereof is derived from a human antibody,preferably produced from a nucleic acid sequence encoding a humanantibody.

The terms “human antibody” and “humanized antibody” are used herein todescribe an antibody of which all portions of the antibody molecule arederived from a nucleic acid sequence encoding a human antibody. Suchhuman antibodies are most desirable for use in antibody therapies, assuch antibodies would elicit little or no immune response in the humanpatient.

The term “chimeric antibody” is used herein to describe an antibodymolecule as well as antibody fragments, as described above in thedefinition of the term “humanized antibody.” The term “chimericantibody” encompasses humanized antibodies. Chimeric antibodies have atleast one portion of a heavy or light chain amino acid sequence derivedfrom a first mammalian species and another portion of the heavy or lightchain amino acid sequence derived from a second, different mammalianspecies. Preferably, the variable region is derived from a non-humanmammalian species and the constant region is derived from a humanspecies. Specifically, the chimeric antibody is preferably produced froma nucleotide sequence from a non-human mammal encoding a variable regionand a nucleotide sequence from a human encoding a constant region of anantibody.

By “binds specifically” is meant high avidity and/or high affinitybinding of an antibody to a specific polypeptide. Antibody binding toits epitope on a specific polypeptide is stronger than binding of thesame antibody to any other epitope, particularly those which may bepresent in molecules in association with, or in the same sample, as thespecific polypeptide of interest. Antibodies that bind specifically to apolypeptide of interest may be capable of binding other polypeptides ata weak, yet detectable, level, e.g. 10% or less of the binding shown tothe polypeptide of interest. Such weak binding, or background binding,is readily discernible from the specific antibody binding to thecompound or polypeptide of interest, e.g. by use of appropriatecontrols.

By “detectably labeled antibody”, “detectably labeled anti-EGF” or“detectably labeled anti-EGF fragment” is meant an antibody (or antibodyfragment that retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels may be selected from a variety of such labels known inthe art, but normally are radioisotopes, fluorophores, enzymes, e.g.horseradish peroxidase, or other moieties or compounds that either emita detectable signal (e.g. radioactivity, fluorescence, color) or emit adetectable signal after exposure of the label to its substrate. Variousdetectable label/substrate pairs (e.g. horseradishperoxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin),methods for labeling antibodies, and methods for using labeledantibodies to detect an antigen are well known in the art (for example,see Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Therapeutic Methods

The present invention provides a method of treating pulmonaryhypersecretion by administering therapeutic amounts of EGF-Rantagonists. Any disease and particularly and pulmonary diseasecharacterized by hypersecretion of mucus or accumulation of pathologicallevels of mucus may be treated by the methods described herein. Examplesof pulmonary hypersecretory diseases that may be treated by this methodinclude, but are not limited to, chronic obstructive lung diseases, suchas chronic bronchitis, inflammatory diseases such as asthma,bronchiectasis, pulmonary fibrosis, COPD, diseases of nasalhypersecretion, e.g. nasal allergies, and other hypersecretory diseases.Genetic diseases such as cystic fibrosis, Kartagener syndrome,alpha-1-antitrypsin deficiency, familial non-cystic fibrosis mucusinspissation of respiratory tract, are intended to be included as well.

Antagonists that directly target EGF or EGF-R are preferred. However,one of skill in the art will appreciate that any factor or cell involvedin the biological cascade that results in EGF-R promoting goblet cellproliferation may be targeted for inhibition, e.g. TGF-α antagonists.Without being bound by theory, a cascade begins during an inflammatoryresponse when cells such as mast cells or neutrophils release TNF-α,which then promotes EGF-R expression. Stimulation of EGF-R, e.g. by itsligand EGF, in turn triggers goblet cell proliferation. Thus, any cellsor factors involved in the cascade, such as in the TNF-α pathway, may betargeted for antagonist activity.

The EGF-R antagonist administered in the therapeutic method may be inany form. By way of example, the EGF-R antagonist may be in the form ofa small molecule (i.e., antisense oligonucleotide, tyrosine kinaseinhibitor, etc.), antibodies or portion of antibodies that bind to EGF,TGFα or EGF-R.

Small Molecule EGF-R Antagonists

Tyrosine kinase inhibitors that act on the EGF receptor, and that areselective for the EGF-R, are known in the art, and may be used in thesubject methods. Examples are described above, and of such may includeBIBX1522 (Boehringer Ingelheim, Inc., Ingelheim, Germany); CGP59326B(Novartis Corporation, Basel, Switzerland); 4-aminoquinazoline EGF-Rinhibitors (described in U.S. Pat. No. 5,760,041); substituted styrenecompounds which can also be a naphthalene, an indane or a benzoxazine;including nitrile and molononitrile compounds (described in U.S. Pat.No. 5,217,999); the inhibitors disclosed in U.S. Pat. No. 5,773,476;potato carboxypeptidase inhibitor (PCI), a 39-amino acid proteaseinhibitor with three disulfide bridges, (Blanco-Aparicio et al. (1998) JBiol Chem 273(20):12370-12377); bombesin antagonist RC-3095 (Szepeshaziet al. (1997) Proc Natl Acad Sci USA 94:10913-10918) etc. Other tyrosinekinase inhibitors include quinazolines, such as PD 153035,4-(3-chloroanilino)quinazoline, or CP-358,774, pyridopyrimidines,pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261and CGP 62706, and pyrazolopyrimidines (Shawn and Shawver, supra.),4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines (Traxier et al. (1996) J.Med. Chem 39:2285-2292), curcumin (Korutla et al. (1994) Biochim BiophysActa 1224:597-600); (Laxmin arayana (1995), Carcinogen 16:1741-1745);etc.

Preferred tyrosine kinase inhibitors are selective for EGF receptor, ie.the EGF-R is inhibited to a greater degree than other cell surfacereceptors having tyrosine kinase activity. Selectivity is enhanced bythe methods of formulation and drug delivery, e.g. where the inhibitoris preferentially delivered to inflamed airways, etc.

Typical dosages for systemic administration range from 0.1 μg to 100milligrams per kg weight of subject per administration. Those of skillwill readily appreciate that dose levels can vary as a function of thespecific compound, the severity of the symptoms and the susceptibilityof the subject to side effects. Some of the specific compounds are morepotent than others. Preferred dosages for a given compound are readilydeterminable by those of skill in the art by a variety of means. Apreferred means is to measure the physiological potency of a givencompound, for example with the in vitro and in vivo tests describedherein.

Antibodies as EGF-R Antagonists

Antibodies as EGF-R antagonists are of particular interest (e.g.Viloria, et al., American Journal of Pathology 151:1523). Antibodies toEGF or EGF-R are produced by immunizing a xenogeneic immunocompetentmammalian host, including murine, rodentia, lagomorpha, ovine, porcine,bovine, etc. with EGF or EGF-R or portions thereof. Preferably human EGFor EGF-R or portions thereof are used as the immunogen. The choice of aparticular host is primarily one of convenience. Immunizations areperformed in accordance with conventional techniques, where theimmunogen may be injected subcutaneously, intramuscularly,intrapentoneally, intravascularly, etc. into the host animal. Normally,from about 1.0 mg/kg to about 10 mg/kg of EGF or EGF-R intraperitoneallyevery other day will be used as an immunogen. The injections may be withor without adjuvant, e.g. complete or incomplete Freund's adjuvant,specol, alum, etc. After completion of the immunization schedule, theantiserum may be harvested in accordance with conventional ways toprovide polyclonal antisera specific for EGF or the EGF-R.

Either monoclonal or polyclonal antibodies, preferably monoclonalantibodies, are produced from the immunized animal. Polyclonal antiseramay be harvested from serum by conventional methods from the animalsafter completion of the immunization schedule. For production ofmonoclonal antibodies, lymphocytes are harvested from the appropriatelymphoid tissue, e.g. spleen, draining lymph node, etc., and fused withan appropriate fusion partner, usually a myeloma line, producing ahybridoma secreting a specific monoclonal antibody. Screening clones ofhybridomas for the antigenic specificity of interest is performed inaccordance with conventional methods.

Of particular interest are antibodies, preferably monoclonal antibodies,that bind to EGF-R or EGF so as to inhibit binding of EGF to EGF-R, e.g.an antibody that specifically binds to the extracellular domain of EGF-Rthereby preventing binding of EGF. Such antibodies may be made byconventional methodology described above, or are commercially available.Examples of antibodies that would function as an EGF antagonist include,but are not limited to, the neutralizing anti-EGF-R monoclonal antibodyC225 (Kawamoto et al. (1983) Proc. Nat'l. Acad. Sci. (USA) 80:1337-1341;Petit et al. (1997) J. Path. 151:1523-153, produced by ImClone SystemsNew York, N.Y.) and the anti-EGF-R monoclonal antibody EMD55900 (alsocalled Mab 425), (Merck, Darmstadt, Germany).

The subject antibodies may be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequencesencoding the variable region of the heavy chain and the variable regionof the light chain are ligated to a spacer encoding at least about 4amino acids of small neutral amino acids, including glycine and/orserine. The protein encoded by this fusion allows assembly of afunctional variable region that retains the specificity and affinity ofthe original antibody.

Methods of humanizing antibodies are known in the art. The humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example, InternationalPatent Applications WO 90/10077 and WO 90/04036). Alternatively, theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the frameworkresidues with the corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isalso known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The chimeric, humanizedantibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

An individual having a hypersecretory mucus disease may initially beadministered amounts of EGF-R antagonist in the range of about 20milligrams (mg) to about 400 mg per kilogram weight of patient twicedaily, e.g. by inhalation.

Antisense Molecules as EGF-R Antagonists

In another embodiment, the subject therapeutic agents are antisensemolecules specific for human sequences coding for EGF or EGF-R. Theadministered therapeutic agent may be antisense oligonucleotides,particularly synthetic oligonucleotides having chemical modificationsfrom native nucleic acids, or nudeic acid constructs that express suchanti-sense molecules as RNA. The antisense sequence is complementary tothe mRNA of the targeted EGF or EGF-R genes, and inhibits expression ofthe targeted gene products (see e.g. Nyce et al. (1997) Nature 385:720).Antisense molecules inhibit gene expression by reducing the amount ofmRNA available for translation, through activation of RNAse H or sterichindrance. One or a combination of antisense molecules may beadministered,where a combination may comprise multiple differentsequences from a single targeted gene, or sequences that complementseveral different genes.

A preferred target gene is EGF-R or EGF. The gene sequence may beaccessed through public databases (human epidermal growth factor,Genbank accession no. K01166; human mRNA for precursor of epidermalgrowth factor receptor, Genbank accession no. X00588). Generally, theantisense sequence will have the same species of origin as the animalhost.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where the vector isintroduced and expressed in the targeted cells. The transcriptionalinitiation will be oriented such that the antisense strand is producedas an RNA molecule. The anti-sense RNA hybridizes with the endogenoussense strand mRNA, thereby blocking expression of the targeted gene. Thenative transcriptional initiation region, or an exogenoustranscriptional initiation region may be employed. The promoter may beintroduced by recombinant methods in vitro, or as the result ofhomologous integration of the sequence into a chromosome. Many strongpromoters that are active in muscle cells are known in the art,including the β-actin promoter, SV40 early and late promoters, humancytomegalovirus promoter, retroviral LTRs, etc.

Transcription vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained incells, usually for a period of at least about one day, more usually fora period of at least about several days.

Alternatively, in a preferred embodiment, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally befrom about 7 to 500, usually from about 12 to 50 nucleotides, moreusually from about 20 to 35 nucleotides, where the length is govemned byefficiency of inhibition, specificity, including absence ofcross-reactivity,and the like. It has been found that shortoligonucleotides, of from 7 to 8 bases in length, can be strong andselective inhibitors of gene expression (see Wagner et al (1996) NatureBiotechnology 14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence. It hasbeen shown that the 5′ region of mRNA is particularly susceptible toantisense inhibition. However, recent evidence indicates analysis ofmRNA secondary structure may be important in accessibility of sites toinhibition. Selection of a specific sequence for the oligonucleotide mayuse an empirical method, where several candidate sequences are assayedfor inhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al (1993) supra. and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiesterstructure, in order to increase their intracellularstability and binding affinity. A number of such modifications have beendescribed in the literature, which alter the chemistry of the backbone,sugars or heterocyclic bases.

Oligonucleotides may additionally comprise a targeting moiety thatenhances uptake of the molecule by cells. The targeting moiety is aspecific binding molecules, e.g. an antibody or fragment thereof thatrecognizes molecules present on the surface of lung epithelial cells,particularly epithelial cells containing EGF-R.

Bispecific antibodies, chimeric antibodies and single chain antibodiesare known in the art. Suitably prepared non-human antibodies can behumanized in various ways. Linkage between the oligonucleotide andtargeting moiety may use any conventional method, for example bydisulfide, amide or thioether bonds, depending on the chemistry of theoligonucleotide backbone. Preferably, the linkage will be cleaved insidethe cell to liberate the oligonucleotide.

Oligonucleotides can be conjugated to hydrophobic residues, e.g.cholesterol, to protect from nucleases and to improve transport acrosscell membranes. Alternatively, conjugation to poly-L-lysine or otherpolyamines may also enhance delivery to the cell. A further modificationthat can be made is the addition of an intercalating component, such asacridine, capable of intercalating into the target mRNA and stabilizingthe resultant hybrid. Antisense oligonucleotides may be transfected incombination with an enzyme(s) that will degrade antisense-mRNA complexesin the cell, e.g. RNase-H. Any protein or enzyme that can preferentiallydegrade or sequester the antisense-mRNA duplex may be similarly useful.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995) Nucl. Acids Res 23:4434-42). Examples of oligonucleotideswith catalytic activity are described in WO 9506764. Conjugates ofanti-sense oligonucleotides with a metal complex, e.g. terpyridylCu(II),capable of mediating mRNA hydrolysis are described in Bashkin et al.(1995) Appl Biochem Biotechnol 54:43-56.

Pharmaceutical Formulations

EGF-R antagonists may be provided in solution or in any otherpharmacologically suitable form for administration, such as a liposomesuspension. The appropriate antibodies or other form of anti-EGF areformulated for administration in a manner customary for administrationof such materials. Typical formulations are those provided inRemington's Pharmaceutical Sciences, latest edition, Mack PublishingCompany, Easton, Pa. The route of administration will be selected basedon the compound being administered, the status of the patient anddisease that is being treated. Where there is hypersecretion of mucus, acompound may be administered through different routes depending on theseverity of the disease, e.g. emergency situations may require i.v.administration, acute but not life threatening situation may be treatedorally, while chronic treatment can be administered by aerosol.

For therapeutic use in nasal and airway diseases, local delivery ispreferred. Delivery by inhalation or insufflating aerosols provide highlevel concentrations of drug compared to the concentration absorbedsystemically. Alternatively, the EGF antagonist maybe administered byinjection, including intramuscular, intravenous (IV), subcutaneous orperitoneal injection, most preferably IV and local injections. However,other modes of administration may also be used provided means areavailable to permit the EGF-R antagonist to enter the systemiccirculation, such as transmucosal or transdermal formulations, which canbe applied as suppositories, skin patches, or intranasally. Any suitableformulation that effects the transfer of the EGF-R antagonist to thebloodstream or locally to the lungs may properly be used.

For injection, suitable formulations generally comprise aqueoussolutions or suspensions using physiological saline, Hank's solution, orother buffers optionally including stabilizing agents or other minorcomponents. Liposomal preparations and other forms of microemulsions canalso be used. The EGF-R antagonist may also be supplied in lyophilizedform and reconstituted for administration. Transmucosal and transdermaladministrations generally include agents that facilitate passage throughthe mucosal or dermal barrier, such as bile, salts, fusidic acid and itsanalogs, various detergents and the like. Oral administration is alsopossible, provided suitable enteric coatings are formulated to permitthe EGF-R antagonist to survive the digestive tract.

The nature of the formulation will depend to some extent on the natureof the EGF-R antagonist chosen. A suitable formulation is prepared usingknown techniques and principles of formulation well known to thoseskilled in the art. The percentage of EGF-R antagonists contained in aparticular pharmaceutical composition will also depend on the nature ofthe formulation; the percentage of an EGF-R antagonist that is anantibody will typically vary over a wide range from about 1% by weightto about 85% by weight.

There are many delivery methods known in the art for enhancing theuptake of nucleic acids by cells. Useful delivery systems include Sendaivirus-liposome delivery systems (Rapaport and Shai (1994) J. BioL Chem.269:15124-15131), cationic liposomes, polymeric delivery gels ormatrices, porous balloon catheters (as disclosed by Shi et aL. (1994)Circulation 90:955-951; and Shi et al (1994) Gene Therapy 1:408-414),retrovirus expression vectors, and the like.

The use of liposomes as a delivery vehicle is one method of interest foruse with EGF-R antagonists. The liposomes fuse with the cells of thetarget site and deliver the contents of the lumen intracellularly. Theliposomes are maintained in contact with the cells for sufficient timefor fusion, using various means to maintain contact, such as isolation,binding agents, and the like. Liposomes may be prepared with purifiedproteins or peptides that mediate fusion of membranes, such as Sendaivirus or influenza virus, etc. The lipids may be any useful combinationof known liposome forming lipids, including cationic lipids, such asphosphatidylcholine. The remaining lipid will normally be neutrallipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol,and the like. For preparing the liposomes, the procedure described byKato et al (1991) J. BioL Chem. 266:3361 may be used.

In a preferred embodiment, the EGF-R antagonist is encapsulated in asterically stabilized “stealth” liposomes, e.g. pegylated liposomes.When such liposomes are injected i.v., they remain in the circulationfor long periods. Postcapillary venular gap junctions open during airwayinflammation and allow fluid accumulation and permit molecules, e.g.complement, kininogen, to enter tissues, initiating inflammatorycascades. Such inflammation allows liposomes and their contents to bedeposited selectively in the inflamed tissue (Zhang et al (1998) PharmRes 15:455-460).

EGF-R antagonists may be administered to the afflicted patient by meansof a pharmaceutical delivery system for the inhalation route. Thecompounds may be formulated in a form suitable for administration byinhalation. The pharmaceutical delivery system is one that is suitablefor respiratory therapy by topical administration of EGF-R antagoniststhereof to mucosal linings of the bronchi. This invention can utilize asystem that depends on the power of a compressed gas to expel the EGF-Rantagonists from a container. An aerosol or pressurized package can beemployed for this purpose.

As used herein, the term “aerosol” is used in its conventional sense asreferring to very fine liquid or solid particles carries by a propellantgas under pressure to a site of therapeutic application. When apharmaceutical aerosol is employed in this invention, the aerosolcontains the therapeutically active compound, which can be dissolved,suspended, or emulsified in a mixture of a fluid carrier and apropellant. The aerosol can be in the form of a solution, suspension,emulsion, powder, or semi-solid preparation. Aerosols employed in thepresent invention are intended for administration as fine, solidparticles or as liquid mists via the respiratory tract of a patient.Various types of propellants known to one of skill in the art can beutilized. Examples of suitable propellants include, but is not limitedto, hydrocarbons or other suitable gas. In the case of the pressurizedaerosol, the dosage unit may be determined by providing a value todeliver a metered amount.

The present invention can also be carried out with a nebulizer, which isan instrument that generates very fine liquid particles of substantiallyuniform size in a gas. Preferably, a liquid containing the EGF-Rantagonists is dispersed as droplets. The small droplets can be carriedby a current of air through an outlet tube of the nebulizer. Theresulting mist penetrates into the respiratory tract of the patient.

A powder composition containing EGF-R antagonists or analogs thereof,with or without a lubricant, carrier, or propellant, can be administeredto a mammal in need of therapy. This embodiment of the invention can becarried out with a conventional device for administering a powderpharmaceutical composition by inhalation. For example, a powder mixtureof the compound and a suitable powder base such as lactose or starch maybe presented in unit dosage form in for example capsular or cartridges,e.g. gelatin, or blister packs, from which the powder may beadministered with the aid of an inhaler.

Combination therapies may be used to treat hypersecretory pulmonarydisease. In particular, EGF-R antagonists may be combined withconventional treatment for alleviation of hypersecretion, such asbronchiodilators, corticosteroids, expectorants, mucolytic agents andthe like to facilitate mucociliary clearance.

Depending on the condition of the patient, it may be preferable todelivery a formulation of the present invention by injection (e.g.,intravenous) or by inhalation. Patients which have large amounts ofmucus in the lungs cannot, in general, be treated initially byinhalation. This is due to the fact that the patient's lungs aresufficiently obstructed that inhaling aerosolized formulation into thelungs may not be particularly effective. However, after treating byinjection or, alternatively, for long term maintenance or in situationswhere the patient's lungs are not severely obstructed, administration byinhalation is preferred. Administration by inhalation is preferredbecause smaller doses can be delivered locally to the specific cellswhich are most in need of treatment. By delivering smaller doses, anyadverse side effects are eliminated or substantially reduced. Bydelivering directly to the cells which are most in need of treatment,the effect of the treatment will be realized more quickly.

There are several different types of inhalation methodologies which canbe employed in connection with the present invention. Antagonists of thepresent invention can be formulated in basically three different typesof formulations for inhalation. First, antagonists of the invention canbe formulated with low boiling point propellants. Such formulations aregenerally administered by conventional meter dose inhalers (MDI's).However, conventional MDI's can be modified so as to increase theability to obtain repeatable dosing by utilizing technology whichmeasures the inspiratory volume and flow rate of the patient asdiscussed within U.S. Pat. Nos. 5,404,871 and 5,542,410.

Alternatively, the agonists of the present invention can be formulatedin aqueous or ethanolic solutions and delivered by conventionalnebulizers. However, more preferably, such solution formulations areaerosolized using devices and systems such as disclosed within U.S. Pat.Nos. 5,497,763; 5,544,646; 5,718,222; and 5,660,166.

Lastly, agonist compounds of the present invention can be formulatedinto dry powder formulations. Such formulations can be administered bysimply inhaling the dry powder formulation after creating an aerosolmist of the powder. Technology for carrying such out is described withinU.S. Pat. No. 5,775,320 issued Jul. 7, 1998 and U.S. Pat. No. 5,740,794issued Apr. 21, 1998.

With respect to each of the patents recited above, applicants point outthat these patents cite other publications in intrapulmonary drugdelivery and such publications can be referred to for specificmethodology, devices and formulations which could be used in connectionwith the delivery of agonists of the present invention. Further, each ofthe patents are incorporated herein by reference in their entirety forpurposes of disclosing formulations, devices, packaging and methodologyfor the delivery of agonist formulations of the present invention.

Screening Assays

Candidate Drugs

Screening assays may be used to identify bioactive candidate agents thatare EGF antagonists. Of particular interest are screening assays foragents that have a low toxicity for human cells. A wide variety ofassays may be used for this purpose, including labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, and the like. The purified EGF orEGF-R protein may also be used for determination of three-dimensionalcrystal structure, which can be used for modeling intermolecularinteractions, transporter function, etc.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or inhibiting thephysiological function of EGF or EGF-R. Generally, a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of hypersecretory disease in formulations described herein.Depending upon the manner of introduction, the compounds may beformulated in a variety of ways described herein. The concentration oftherapeutically active compound in the formulation may vary from about0.1-100% by weight.

Dosage Regime

The appropriate dosage level will also vary depending on a number offactors including the nature of the subject to be treated, theparticular nature of the hypersecretory condition to be treated and itsseverity, the nature of the EFGR antagonist used as active ingredient,the mode of administration, the formulation, and the judgement of thepractitioner. For example, when antibodies are administered bythemselves such as anti-EGF or EGF-R in an injectable formulation, thedosages will be in the range of 20 mg/kg to about 40 mg/kg at a singledosage. Repeated administration over a period of days may be required oradministration by intravenous means may be continuous. For chronicconditions, administration may be continued for longer periods asnecessary.

Efficacy of the dosing regime will be determined by assessing forimproved lung function in the patient. This assessment may includeviscoelasticity measurements of sputum, improvements in pulmonaryfunction, including improvements in forced exploratory volume of sputumand maximal midexpiratory flow rate. The aforementioned therapeuticregime can be given in conjunction with adjunct therapies such asantibiotics, DNAse I or other current therapies for the treatment ofhypersecretory pulmonary disease. If antibiotics are co-administered aspart of the patient's therapy, bacterial quantitation following therapycan be included to assess the efficacy of the treatment by decreasedbacterial growth, indicating decreased viscosity of mucus or sputum andincrease of the mucus or sputum lung clearance.

Pulmonary function tests, as well as diagnostic tests for the clinicalprogression of pulmonary hypersecretory disease, are known to thoseindividuals with skill in this art. Standard pulmonary function testsinclude airway resistance (AR); forced vital capacity (FVC); forcedexpiratory volume in 1 second (FEV(1)); forced midexpiratory flow; andpeak expiratory flow rate (PEFR). Other pulmonary function tests includeblood gas analysis; responses to medication; challenge and exercisetesting; measurements of respiratory muscle strength; fibro-optic airwayexamination; and the like. Some basic procedures for studying theproperties of mucus include rheology, e.g. with the use of a magneticmicrorheometer; adhesivity to characterize the forces of attractionbetween an adherent surface and an adhesive system by measuring thecontact angle between a mucus drop and a surface. Mucus transport bycilia can be studied using conventional techniques, as well as directmeasurement, i.e. in situ mucus clearance. Transepithelial potentialdifference, the net result of the activity of the ion-transport systemof the pulmonary epithelium, can be measured using appropriatemicroelectrodes. Quantitative morphology methods may be used tocharacterize the epithelial surface condition.

The patient to be treated can be a primate, such as a human, or anyother animal exhibiting the described symptoms. While the method of theinvention is especially adapted for the treatment of a human patient, itwill be understood that the invention is also applicable to veterinarypractice.

In Vitro Screening Assay

In another embodiment of this invention, in vitro assays are used toassess the therapeutic potential of candidate agents to inhibit gobletcell proliferation, i.e. whether such agents are active as an EGFantagonist. Generally, such assays will comprise the following steps:(i) contacting an in vitro model of goblet cell proliferation with EGFor the functional equivalent thereof; (ii) subsequently contacting thein vitro model with a candidate agent; and (iii) assessing goblet cellproliferation, wherein an inhibition of goblet cell proliferation isindicative of the candidate agent's therapeutic potential.

The assay is preferably carried out with two controls where a secondcell group is not contacted with any compound and a third is contactedwith EGF but not the candidate agent. Comparisons are then made todetermine the degree of effect of EGF and the candidate agent on thecells.

Any in vitro model of goblet cell proliferation may be used. By way ofexample, rat tracheal cells can be isolated and maintained in culture asdescribed in Guzman et al. (1995) 217:412-419. Briefly, the rat trachealcells are plated onto collagen gel coated semipermeable membranes,initially cultured submerged in media, and subsequently maintained withan air/liquid interface. Examples of in vitro cells include primaryhuman bronchial cells (available from Clonetics, San Diego); NCI-H292cells (ATCC CRL-1848); and A431 cells (ATCC CRL-1555).

The in vitro culture is contacted with EGF and with a candidate agent.The candidate agent may be contacted with the culture prior to,concurrently with, or subsequently to the addition of EGF depending onthe endpoint to be assessed and the nature of the candidate agent. Thecultured cells are assessed for inhibition of goblet cell proliferationrelative to controls.

A variety of molecular or biochemical markers may be used to assessgoblet cell proliferation. Examples of molecular or biochemical markersthat may be used include, but are not limited to, gene expression orprotein expression characteristic of goblet cells. Certain mucin genes,e.g. MUC5B (Desseyn et al. (1997) J. Biol. Chem. 272:3168-3178) areexpressed in the airway, and have a gene product highly represented inmucus. Expression of mucin genes provides a suitable marker fordetermining production of mucus.

Mucin gene expression may be assessed by conventional technology such asnorthern blot analysis, polymerase chain reaction (PCR), examination ofthe mucin gene promoter, or in situ analysis. Alternatively mucinproteins are assessed by conventional methodology, such as western blotanalysis, ELISA, immunochemistry and the like, using detectably labeledantibodies. Morphological criteria may also be used to determine thepresence or absence of goblet cells in the culture; such as staining formucins using Alcian blue/PAS staining (Lou et al. (1998) Am. J. Respir.Crit. Care Med. 157:1927-1934). Antibodies to mucins can be examinedusing ELISA assays. Because stimulation of EGF-R by a ligand, e.g. EGF,TGF-α, induces phosphorylation of a specific EGF receptor kinase andresults in goblet cell production, EGF-R phosphorylation can be measuredas a reflection of goblet cell induction (Donato et aL (1984) J. Biol.Chem. 264:20474-20481).

A decrease in the molecular on biochemical markers associated withgoblet cell proliferation is indicative of the therapeutic potential ofthe antagonist.

In vivo Models

In yet another embodiment of the invention, in vivo animal models areused to assess the therapeutic potential of candidate agents to inhibitgoblet cell proliferation. Generally the assay comprises the steps of:(i) creating an animal model of hypersecretory pulmonary disease byinducing EGF-R expression; (ii) stimulating the induced EGF-R to producemucin producing goblet cells; (iii) treating with a candidate agent; and(iv) assessing goblet cell proliferation or mucus secretion, wherein aninhibition of goblet cell proliferation or mucus secretion is indicativeof the candidate agent's therapeutic potential.

Any in vivo model of hypersecretory pulmonary disease may be used. Byway of example an asthmatic mouse model, as described in Temann et al.(1997) Am. J. Respir. Cell. Biol. 16:471-478, and as shown in theexamples provided herein. Alternatively, a rat model can be used, asdescribed by Takeyama et al. (1998) Am. J. Physiol. Examples of otheranimal models that may be used include, but are not limited to Guineapigs (a species that expresses goblet cells constitutively) and rats.

The lung tissue or tracheal tissue of the animal models may be assessedby the same molecular and biochemical markers described for the in vitromodel. A decrease in goblet cell proliferation is indicative of thetherapeutic potential of the EGF-R antagonist.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXPERIMENTAL EXAMPLE 1 The EGF System Regulates Mucin Production inAirways

Goblet cell hyperplasia occurs in various hypersecretory diseases ofairways, but because the underlying mechanisms are unknown, no effectivetherapy exists. In healthy airways, goblet cells are few, but inhypersecretoryairway diseases, goblet cell hyperplasia occurs. A humanbronchial (NCI-H292) cell line was studied. These cells express EGF-Rconstitutively; EGF-R gene expression was further stimulated by tumornecrosis factor alpha (TNFα). EGF-R ligands increased the synthesis ofmucins, and this effect was increased by co-incubation with TNFα.

Airway epithelial cells of pathogen-free rats expressed little EGF-Rprotein, but intratracheal instillation of TNFα (200 ng) induced EGF-Rin basal, pre-goblet, and goblet cells, but not in ciliated cells; TNFα,EGF, or TGFα alone did not induce goblet cell production. However,instillation of TNFα, followed by EGF-R ligands resulted in an increasednumber of goblet and pre-goblet cells and a striking increase in Alcianblue/PAS-positive staining (reflecting mucous glycoconjugates) and mucinMUC5 gene expression. In sensitized rats, ovalbumin resulted in gobletcell production and EGF-R expression in airway epithelium. In NCI-H292cells, in rats stimulated by TNFα followed by EGF-R ligands, and in theasthma model in rats, pretreatment with EGF-R tyrosine kinase inhibitor(BIBXL 1522) prevented goblet cell production in airways. These findingsdemonstrate a role for inhibitors of the EGF-R cascade in hypersecretorydiseases of airways.

METHODS

IN VITRO STUDIES.

Cell culture. A human pulmonary mucoepidermoid carcinoma cell line,NCI-H292 cells, were grown in RPMI 1640 medium containing 10% fetalbovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml) at 37° C.in a humidified 5% CO₂ water-jacketed incubator. When confluent, cellswere incubated with EGF (recombinant human EGF, 25 ng/ml, Genzyme,Cambridge, Mass.), TGFα (recombinant human TGFα, 25 ng/ml, Genzyme),TNFα (recombinant human TNFα, 20ng/ml, Genzyme), EGF (25 ng/ml) plusTNFα (20ng/ml) or TGFα (25 ng/ml) plus TNFα (20 ng/ml) for 12 h, 24 h or48 h. In inhibition studies with an EGF-R tyrosine kinase inhibitor,BIBX1522 (10 μg/ml, generously provided by Boehringer Ingelheim Inc.,Ingelheim, Germany), cells were pretreated with BIBX1522 30 min beforeadding growth factors. After incubation, cells grown in a T-75 flaskwere used for total RNA extraction or protein extraction, and 8-chamberslides were used for Alcian blue/PAS-staining to visualize mucins.

Western blotting. Cells grown in T-75 flasks were lysed and scraped withPBS containing 1% Triton X, 1% sodium dioxycolate and PMSF (10 mg/ml).Total amount of protein was estimated by BCA protein assay reagent(Pierce, Rockford, Ill.). Cell lysates were boiled with Tricine samplebuffer and 2% βME at 95° C. Proteins were separated by SDS-PAGE in 8%acrylamide gels. The resulting gels were equilibrated in the transferbuffer: 25 mM Tris-HCI, 192 mM glycine, 20% (vol/vol) methanol, pH 8.3.The proteins were then transferred electrophoretically to nitrocellulosemembranes. The membranes were then incubated for 1 h in 5% fat-free skimmilk in PBS containing 0.05% Tween 20. Then the membranes were incubatedwith monoclonal mouse anti-EGF-R antibody (1:100) at 4° C. overnight.Bound antibody was visualized according to standard protocols foravidin-biotin-alkaline phosphatase complex method (ABC kit, VectorLaboratories). As a positive control for EGF-R, cell lysates from A431cells were used (20).

Immunocytochemical localization of EGF-R in NCI-H292 cells. Cells grownon 8-chamber slides were fixed with 4% paraformaldehyde for 1 h. Tostain for EGF-R, PBS containing 0.05% Tween 20, 2% normal goat serum and2 mM levamisole was used as diluent for the antibody. Sections wereincubated with mouse monoclonal antibody to EGF-R (1:250) overnight at4° C., and then washed 3 times with PBS to remove excess primaryantibody. Cells were then incubated with biotinylated horse anti-mouseimmunoglobulin (Vector Laboratories, Burlingame, Calif.) at 1:200dilution for 1 h at room temperature. Bound antibody was visualizedaccording to standard protocols for avidin-biotin-alkaline phosphatasecomplex method (ABC kit, Vector Laboratories, Burlingame, Calif.).

Probes. EGF-R mRNA expression was determined using the linearizedpTRI-EGF-R-human probe template (Ambion, Austin, Tex.). This probecontains a 360 bp cDNA fragment of the human EGF-R gene, which spansexons 12-14. MUC5 gene expression was determined using human MUC5ACprobe, which contains a 298 bp cDNA fragment of human MUC5AC gene(generously provided by Dr. Carol Basbaum).

Northem bloffing. Total RNA was extracted from NCI-H292 cells grown in aT-75 tissue culture flask using Tri-Reagent (Molecular Research Ctr,Cincinnati, Ohio) in each condition. Total RNA (10 μg) waselectrophoresed on 1% agarose/formaldehydegel and transferred to a nylonmembrane (Amersham, Arlington Heights, Ill.) by capillary blotting. Theprobes were labeled with ³²P using the Random Primed DNA labeling kit(Boehringer Mannheim Corp., Indianapolis, Ind.). Blots wereprehybridized at 42° C. for 4 h and then hybridized at 42° C. for 16 hwith ³²P-labeled specific cDNA probe. Hybridization solution contained250 mM Tris-HCI (pH7.5), 5% SDS, 1% BSA, 1% polyvinyl-pyrrolidone, 1%Ficoll, and 0.5% sodium pyrophosphate. After hybridization, themembranes were washed twice with 2 ×SSC with 0.1% SDS for 30 min at roomtemperature, followed by two washes in 2 ×SSC with 0.1% SDS for 30 minat 50° C. and a rinse in 0.1 ×SSC with 0.1% SDS. Membranes were exposedto X-ray film.

In Vivo Studies.

The experimental animal protocol was approved by the Committee on AnimalResearch, University of California San Francisco. Specific pathogen-freemale F344 Fisher rats, weighing 230-250 g (Simonsen Laboratories,Gilroy, Calif.), were maintained in a temperature-controlled (21° C.)room with standard laboratory food and water freely available.

Healthy rats. Rats were anesthetized with methohexital sodium (Brevitalsodium, 50 mg/kg, i.p.; Eli Lilly & Co., Indianapolis, Ind.) and allowedto breathe spontaneously. To determine whether TNFα up-regulates EGF-Rin airways, TNFα (200 ng, 100 μl) was instilled into the trachea and theanimals were euthanized 24 h later. To examine whether EGF or TGFαinduces goblet cells in airway epithelium, EGF (600 ng, 100 μl) or TGFα(rat synthetic TGFα, 250 ng, 100 μl; Sigma, St Louis, Mich.) wasinstilled into the trachea either alone or 24 h after the instillationof TNFα (200 ng, 100 μl), and the animals were euthanized 48 h later. Ineach study, sterile PBS (100 μl) was instilled into the trachea ascontrol. To confirm whether mucin production occurred via activation ofEGF-R, we examined the effect of an EGF-R tyrosine kinase inhibitor,BIBX1522 (dose estimated from studies using the inhibitorto preventcancer growth). Rats were pretreated with BIBX1522 (3, 10 or 30 mg/kg,i.p.), 1 h before and 24 h after instillation of TGFα. The trachea andlungs were removed for examination 48 h after the instillation of TGFα.ps Sensitized Rats

Sensitization. Rats were sensitized on days 0 and 10 withintraperitoneal injections of ovalbumin (10 mg, grade V; Sigma, St.Louis, Mo.), complexed with 100 mg of aluminum hydroxide in 0.5 ml ofsterile saline. Rats then rested for 10 days. On day 20, ovalbumin wasdelivered directly into the trachea; animals were challenged with 100 μlof 0.1% ovalbumin in saline by intratracheal instillation three times(days 20, 22 and 24). Rats were euthanized either without challenge (day20), or 48 h after the third challenge (day 26). This procedure inducedgoblet cell metaplasia. To block the goblet cell hyperplasia, sensitizedrats were pretreated with an EGF-R tyrosine kinase inhibitor, BIBX 1522.On days of ovalbumin challenge (days 20, 22 and 24), sensitized ratswere pretreated with BIBX 1522 (10 mg/kg, i.p., 1 h before thechallenge) and then BIBX1522 was also instilled into the tracheatogether with ovalbumin (BIBX 1522, 10⁻⁵M, 100 μl). BIBX 1522 was alsoinjected i.p. every 24 h until the day before the rats were euthanized.After the animals were euthanized, the trachea was removed 48 h afterthe third challenge.

Tissue preparation. At preselected times during anesthesia, the systemiccirculation was perfused with 1% paraformaldehyde in DEPC-treated PBS ata pressure of 120 mmHg. The trachea was then removed and placed in 4%paraformaldehyde for 24 h. After fixation, trachea and lungs wereembedded in either JB-4 plus monomer solution A for cell analysis orO.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, Calif.) forimmunohistochemistry and in situ hybridization. The embedded tissueswere cut as cross sections (4 mm thick) and placed on slides.

Cell analysis. We counted the total number of epithelial cells bycounting epithelial cell nuclei over 2 mm of the basal lamina with anoil immersion objective lens (×1000 magnification). The linear length ofthe basal lamina under each analyzed region of epithelium was determinedby tracing the contour of the digitized image of the basal lamina. Afterinstillation of stimuli, “developing” goblet cells form. These cellshave Alcian blue/PAS-positive granules, but the size of granules issmall, and the number of cytoplasmic granules is few. We call these“developing” goblet cells “pre-goblet cells”, a stage before cellsbecome mature goblet cells. Goblet cells are tall, cuboidal, goblet tolow columnar in shape, with abundant Alcian blue/PAS-stained granulesfilling most of the cytoplasm between the nucleus and the luminalsurface. Pre-goblet cells are defined as cells with smallermucus-stained areas (<⅓ height in epithelium from basement membrane toluminal surface) or with sparsely and lightly Alcian blue/PAS-stained,small granules. Ciliated cells are recognized by their ciliated borders,lightly stained cytoplasm, and large round nuclei. Non-granulatedsecretory cells are columnar in shape and extend from the lumen to thebasal lamina. The cytoplasm stains light pink color, and a few tinyPAS-positive and Alcian blue-negative granules are observed in thecytoplasm. Basal cells are small flattened cells with a large nucleus,located just above the basal lamina but not reaching the airway lumen.

Quantification of goblet cell production. Goblet cell production, wasdetermined by the volume density of Alcian blue/PAS-stainedmucosubstances on the mucosal surface epithelium using a semi-automaticimaging system described elsewhere (Weber et al. (1984) Science224:294-297). We measured the Alcian blue/PAS-positive stained area andthe total epithelial area and expressed the data as the percentage ofthe total area stained by Alcian blue-PAS. The analysis was performedwith the public domain NIH Image program (developed at the U.S. NationalInstitute of Health and available from the Internet by anonymous FTPfrom zippy.nimh.gov or on floppy disk from the National TechnicalInformation Service, Springfield, Va., part number PB95-500195GEI).

Immunohistochemical localization of EGF-R in rat epithelium. Thelocalization of EGF-R was examined using immunohistochemical stainingwith an antibody to EGF-R (Calbiochem, San Diego, Calif.) in frozensections of rat trachea. After perfusion with 1% paraformaldehyde inPBS, tissues were placed in 4% paraformaldehyde in PBS for 1 h and thenremoved in 30% sucrose for cryoprotection overnight. Tracheas wereembedded in O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance,Calif.) and frozen. Frozen sections (5 μm) were cut and placed on glassslides (Superfrost Plus, Fisher Scientific, Pittsburgh, Pa.).Immunostaining was performed similarly to the in vitro studies.

Probe Preparation. The cDNA for rat MUC5 was generously provided by Dr.Carol Basbaum. A 320 bp cDNA fragment of rat MUC5 was subcloned into theXba/hindlll site of the transcription vector, pBluescript-SK(-)(Stratagene, La Jolla, Calif.). To prepare RNA probes for in situhybridization, this recombinant plasmid containing the rat MUC5 cDNAfragment was linearized and transcribed in vitro with the T7 or T3polymerase to obtain antisense or sense probe, respectively. The probesfor in situ hybridization were generated in the presence of [³⁵S]UTP.After transcription, the cDNA template was digested with DNase, andradiolabeled RNA was purified via a Sephadex G-25 Quick SpinTM Column(Boehringer Mannheim, Indianapolis, Ind.) and precipitated in anethanol/ammonium acetate solution. Before use, RNA probes were washedwith 70% ethanol and diluted in 10 mM DTT.

In Situ Hybrdization. Frozen sections (5 μm) were cut and placed onpositively charged glass slides (Superfrost Plus, Fisher Scientific,Pittsburgh, Pa.,). Sections cut in close proximity were used forhybridization with sense and antisense probes. Alternate sections wereused for Alcian blue/PAS staining. Specimens were refixed in 4%paraformaldehyde, rehydrated in 0.5 ×SSC, and then acetylated intriethanolamine with acetic anhydride. Hybridization was carried outwith 2500-3000 cpm/μl of antisense or sense probe in 50% deionizedformamide, 0.3 M NaCI, 20 mM Tris, 5 mM EDTA, 1 ×Denhardt's solution, 20mM dithiothreitol, 10% dextran sulfate, 0.5 mg/ml yeast tRNA, and 0.5mg/ml sonicated salmon sperm DNA at 55 oC overnight. Posthybridizationtreatment consisted of washes with 2×SSC, 1 mM EDTA, 10 mMβ-mercaptoethanol at room temperature, incubation with RNase solution(20 mg/ml) for 30 min at room temperature, and further washes in0.1×SSC, 1 mM EDTA, 10 mM β-mercaptoethanol at 55° C. for 2 h and thenin 0.5×SSC at room temperature. Specimens were dehydrated, air-dried,and covered with Kodak NBT nuclear track emulsion (Eastman Kodak,Rochester, N.Y.) for autoradiography. After exposure for 7 to 21 d at 4°C., the slides were developed, fixed, and counterstained withhematoxylin (21).

Statistics. All data are expressed as mean ± SEM. One-way analysis ofvariance was used to determine statistically significant differencesbetween groups. Scheffe's F test was used to correct for multiplecomparisons when statistical significances were identified in theanalysis of variance. A probability of less than 0.05 for the nullhypothesis was accepted as indicating a statistically significantdifference.

RESULTS

TNFα Stimulates Production of EGF-R in NCI-H292 Cells. First wedetermined whether NCI-H292 cells express EGF-R constitutively. Westernanalysis of immunoblots identified the presence of EGF-R protein inconfluent cultures of NCI-H292 cells (FIG. 1A, right). Cells wereexamined after becoming confluent. Lysates were electrophoresed in 8%acrylamide gels and blotted with anti-EGF-R antibody. Molecular weightsof marker proteins are reported on the right. A positive control forEGF-R was protein from A431 cells (FIG. 1A, left), which express EGF-Rconstitutively (Weber et al., supra.). Immunocytochemical studies withan anti-EGF-R antibody revealed positive staining, most striking individing cells (FIG. 1B, Immunocytochemical analysis with anti-EGF-Rantibody in cultures of NCI-H292 cells). At confluence, positivestaining was seen, most strongly in dividing cells (arrows, right side).In the absence of the primary antibody, staining was absent (left side).Northern blotting showed that TNFα (20 ng/ml) up-regulated EGF-R geneexpression, an effect that was present at 12 h and increased at 24 h(FIG. 1C, Northern analysis of EGF-R in NCI-H292 cells). Analysis wasperformed on total RNA extracted from confluent cultures incubated withTNFα (20 ng/μl) for 12 or 24 h. The RNA was electrophoresed on aformaldehyde-agarose gel, transferred to a nylon membrane, andhybridized with the ³²P-labeled EGF-R cDNA probe. After hybridization,the membrane was washed and autoradiographed.

EGF-R Ligands Stimulate Expression of Mucous Glycoconjugates and MUC5Gene Expression in NCI-H292 Cells. EGF-R are expressed constitutively inNCI-H292 cells, so we assessed the ability of EGF-R ligands (EGF, TGFα)to induce the production of mucous glycoconjugates (FIG. 2, uppercolumn, Alcian blue/PAS staining of NCI-H292 cells for identification ofmucin glycoproteins). Upper column=incubation of cells withoutinhibitor; lower column incubation in the presence of the EGF-R tyrosinekinase inhibitor BIBX1522 (10 μg/ml). When cells were incubated alone(control), some PAS-positive staining was seen (arrows, upper column);incubation with TNFα (20 ng/ml) alone did not affect the staining;incubation with EGF; (25 ng/ml) or with TGFα (25 ng/ml) increased thePAS-positive staining (arrows); incubation with TNFα plus TGFα increasedmarkedly staining (arrow, upper column).

Some control cells showed staining; incubation with TNFα (20 ng/ml)alone did not affect staining; incubation with either EGF or with TGFα(each at 25 ng/ml) increased PAS-positive staining (arrows); incubationwith TNFα plus TGFα increased the staining much more than either ligandalone. Thus, EGF-R ligands induce mucous glycoconjugates in NCI-H292cells.

To examine MUC5 gene expression, Northern blotting was performed (FIG.3). Total RNA (10 μg) was extracted from the cells, electrophoresed on aformaldehyde-agarose gel, transferred to a nylon membrane, andhybridized with the ³²P-labeled MUC5 cDNA probe. After hybridization,the membrane was washed and autoradiographed. Cultures were obtainedwith medium alone (C), EGF or TGFα (25 ng/ml), TNFα (20 ng/ml), or thecombination of TNFα plus either EGF or TGFα for 12 (upper column) or 24h (lower column) on MUC5 gene expression. Cultures were also obtainedwith TNFα plus either EGF-or TGFα after preincubation with EGF-Rtyrosine kinase inhibitor (BIBX1522; 10 μg/ml; lower column); theinhibitor prevented MUC5 gene expression.

NCI-H292 cells showed some expression in the control state (FIG. 3,lower left column); when the cells were incubated with EGF or TGFα, MUC5gene expression was barely recognized at 12 h but was clearly expressedat 24 h. TNFα alone did not affect MUC5 gene expression, but when TNFαwas added to the cells incubated with EGF-R ligands, MUC5 geneexpression increased markedly above the level caused the EGF-R ligandalone (FIG. 3).

EGF-R Tyrosine Kinase Inhibitor (BIBX 1522) Prevents Expression ofMucous Glycoconjungates and of MUC5 Gene Expression in NCI-H292 Cells.To test the hypothesis that activation of EGF-R receptors induces MUC5gene expression, cells were incubated with an EGF-R tyrosine kinaseinhibitor BIBX1522. When NCI-H292 cells were pretreated with BIBX1522(10 1μg/ml), PAS-positive staining was inhibited in the control state,and the increased staining that occurred with the EGF-R ligands wasmarkedly inhibited (FIG. 2, lower column). On Northern analysis, MUC5gene expression that was markedly increased by the combination of TNFαplus EGF or plus TGFα was completely inhibited by pre-incubation withBIBX1522 (FIG. 3, lower column). These results implicate activation ofEGF-R in the induction of mucin gene and mucous glycoproteins inNCI-H292 cells.

TNFα Stimulates Production of EGF-R in Rats. Pathogen-free rats (whichhave few airway epithelial goblet cells constitutively) were studied,starting with the role of TNFα. In the control state, trachealepithelium contained few EGF-R-positive cells (FIG. 4A, left). However,intratracheal instillation of TNFα (200 ng) induced EGF-R protein invarious cell types in the tracheal epithelium (FIG. 4A, right).EGF-R-positive staining was present in goblet cells (G), pre-gobletcells (P-G), non-granulated secretory cells (S), and basal cells (Ba),but not in ciliated-cells. Thus, TNFα induces EGF-R protein production.

Role of EGF-R Ligands in Production of Mucous Glycoconjugates and MUC5Gene Expression in Rats. In the control state, tracheal epitheliumcontained few goblet and pre-goblet cells. Intratracheal instillation ofEGF-R ligands, EGF (600 ng; not shown) or TGFα (250 ng; Table 1) alonehad no effect on epithelial production of mucous glycoconjugates.However, when TNFα (200 ng) was given first, followed in 24 h by EGF orTGFα (Table 1), and the animals were euthanized 48 h later, Alcianblue/PAS staining was increased markedly, and the numbers of goblet andpre-goblet cells were markedly increased, without a change in the totalnumber of cells or in the number of ciliated cells (Table 1). In situhybridization for MUC5 gene showed no expression in control animals.When TNFα, followed by EGF or TGFα, was instilledintratracheally,expression of MUC5 was visible in the epithelium. Thus,induction of EGF-R alone or stimulation by EGF-R ligands alone wasinsufficient to induce goblet cell metaplasia or the production ofmucous glycoconjugates. However, after the induction of EGF-R by TNFα,instillation of EGF-R ligands stimulated goblet cell metaplasiamarkedly.

TABLE 1 Cell Analysis in tracheal epithelium Ova sensitization Cell typecontrol TGFα TNFα/TGFα i.p. only i.p. + i.t. Goblet 2.8 ± 0.7 5.8 ± 1.228.8 ± 3.4* 5.4 ± 1.5 38.2 ± 6.3* Pre-goblet 7.8 ± 1.3 12.8 ± 1.6  44.8± 3.6* 13.8 ± 1.4  36.0 ± 6.3* Secretory 82.0 ± 2.0  72.2 ± 4.0  40.8 ±2.4* 67.6 ± 7.0  49.8 ± 4.2  Ciliated 49.6 ± 2.0  54.6 ± 2.3  53.2 ±1.8  56.4 ± 3.8  52.4 ± 7.1  Basal 57.8 ± 2.6  56.8 ± 2.3  43.0 ± 3.5 60.2 ± 3.4  59.8 ± 2.9  Indeterminate 1.4 ± 0.5 2.0 ± 0.4 0.8 ± 0.4 1.4± 0.2 2.6 ± 0.5 Total 201.4 ± 2.2  204.2 ± 3.3  211.4 ± 4.8  204.8 ±6.6  238.8 ± 4.4*  % of AB/PAS-stained 2.4 ± 0.8 6.8 ± 1.9 35.8 ± 4.2*7.5 ± 2.9 38.7 ± 6.2* area Effect of mediators and of ovalbuminsensitization on tracheal epithelial cells in rats. Cells were analyzedas described in Methods; five rats per group. Characterization was aidedby Alcian blue (AB)/PAS staining (which stains mucous glycoconjugates).In addition to counting of cells, percent of the total epithelial areaoccupied by AB/PAS-staining was calculated. Control airways and airwaysstimulated by TGFα (250 ng) alone contained few goblet and pre-gobletcells; #there was little staining with AB/PAS. TNFα (200 ng), followedby TGFα, resulted in increased numbers of goblet and pre-goblet cellsand an increase in the area occupied by AB/PAS-stained cells.Sensitization of rats with ovalbumin (OVA) intraperitoneally (ip) had noeffect on cell distribution or on AB/PAS staining, but when OVA wasgiven ip followed by intratracheal (it) instillation of OVA, a strikingincrease in goblet and pre-goblet cells and the percent area occupied by#AB/PAS stain was found.

Ovalbumin Sensitization in Rats Induces EGF-R and Goblet CellProduction. Because death from acute asthma is reported to be due tomucous obstruction of airways, a model of asthma was produced inpathogen-free rats. Injections of ovalbumin (10 mg, ip) on days 0 and 10did not stimulate goblet cell hyperplasia (Table 1). However, when thiswas followed by three intratracheal (i.t.) instillations of ovalbumin(0.1% in 100 μl) on days 20, 22, and 24, and the animals were euthanizedon day 26, the numbers of goblet and pre-goblet cells were increasedmarkedly; the numbers of ciliated and basal cells were unchanged (Table1, right side). Immunohistochemical studies with an anti-EGF-R antibodyshowed no staining in control tracheas. Animals sensitized both i.p. andi.t. showed EGF-R staining (FIG. 4B, left) selectively in cells thatstained positively with AB/PAS (FIG. 4B, right). After 3 intrachealinstillations of ovalbumin (0.1%, 100 ml), EGF-R immunoreactivity wasstrongly expressed in goblet and pre-goblet cells (lower left), the samecells that stained positively with Alcian blue/PAS (lower right). Thus,an ovalbumin model of asthma showed goblet cell proliferation in cellsthat produced EGF-R.

EGF-R Tyrosine Kinase Inhibitor (BIBX1522) Prevents Goblet CellProduction Induced by Instillation of TNFα Plus EGF-R Ligands and byOvalbumin Sensitization in Rats. Because BIBX1522 prevented mucinproduction in cultured cells, the effect of this inhibitorwas examinedin pathogen-free rats. Alcian-blue/PAS staining that was increased bytracheal instillation of TNFα followed by the EGF-R ligand TGFα, wasinhibited in a dose-dependent fashion by pretreatment with BIBX1522(3-30 mg/kg, ip; FIG. 5A). Tracheal instillation of TNFα (to induceEGF-R), followed by the EGF-R ligand TGFα, resulted in striking gobletcell metaplasia.

In rats sensitized with ovalbumin, pretreatment with BIBX1522 (10 mg/kg,ip) inhibited the production of goblet cells completely (evaluated byAlcian blue/PAS staining; FIG. 5B). Animals given ovalbumin i.p. onlyshowed little AB/PAS-positive staining in bronchial epithelium. Animalsfirst sensitized with OVA i.p., followed by three intratracheal (i.t.)instillations of OVA, showed a marked increase in AB/PAS-positivestaining.

These studies indicate that EGF-R, when stimulated by EGF-R ligands,induce goblet cell production in vitro and in vivo, effects due toactivation of EGF-R and which were blocked by an EGF-R tyrosine kinaseinhibitor. In an ovalbumin model of asthma, the inhibitor was alsoeffective in preventing goblet cell production.

In addition to describing a mechanism for inducing goblet cells, presentresults suggest a possible sequence for the evolution of goblet cellproduction based on the expression of EGF-R: Stimulation with TNFαinduced intense staining of non-granulated secretory cells; theirsubsequent activation by EGF-R ligands caused progressive staining formucous glycoconjugates in the cytoplasm, and the cells became“pre-goblet” and then “goblet” cells. Instillation of TNFα followed byEGF-R ligands induced goblet cell production without altering the totalnumber of epithelial cells, suggesting that EGF-R activation promotedselective cell differentiation (not proliferation). The findings suggestthat goblet cells are derived from non-granulated secretory cells thatexpress EGF-R and are stimulated by EGF-R ligands to produce mucins.

In patients who die of acute asthma, goblet cell hyperplasia and mucousplugging are important findings. In a murine model of asthma,sensitization of airways occurs after repeated instillation ofovalbumin, resulting in marked airway goblet cell hyperplasia. We showthat EGF-R, which is not expressed in control airway epithelium, isexpressed in sensitized animals. Cells that stained were pre-goblet andgoblet cells, suggesting that EGF-R was involved in goblet cellproduction. Pretreatment with an EGF-R receptor tyrosine kinaseinhibitor (BIBX1522) prevented airway goblet cell production, confirmingthe role of EGF-R activation in goblet cell production in experimentalasthma.

Present results implicate the EGF-R pathway in goblet cell hyperplasia.Previous studies have shown that various stimuli such as ozone, sulfurdioxide, viruses, lipopolysaccharide, platelet activating factor, andinterleukin-4 up-regulate mucin expression and secretion. The presentinvention provides a mechanism to evaluate the relationship of theseinflammatory stimuli and the EGF-R system.

Asthma serves as an example of the therapeutic strategy of theinvention: Normal human airway epithelium has a ratio of 3-10 ciliatedcells to each goblet cell. In asthma, the number of goblet cells can beequal or exceed ciliated cells; in patients who die in statusasthmaticus, there is a 30-fold increase in the percentage area occupiedby goblet cells compared with the number in patients dying of non-asthmarespiratory diseases. Inhibition of production of goblet cells shouldeliminate this source of hypersecretion. Because the life cycle ofgoblet cells is unknown, the time course of resolution of goblet cellhyperplasia with treatment can not be predicted with precision. In theabsence of further exposure to allergen, goblet cell hyperplasia inpreviously sensitized mice resolved within fifty days, along with othermanifestations of allergic inflammation. Inhibition of EGF-R activationmay inhibit goblet cell hyperplasia much more rapidly, depending on thelife span of goblet cells. Recently, highly selective ATP-competitivetyrosine kinase inhibitors have been reported. EGF-R tyrosine kinaseinhibitors are being evaluated for the treatment of malignanciesassociated with the expression of EGF-R.

Hypersecretion is a major manifestation in many chronic inflammatorydiseases of airways. Presently, there is no effective therapy to relievethe symptoms and to halt the progression of these diseases. Presentfindings provide a mechanism and a strategy for therapy: by inhibitingEGF-R activation, goblet cell production is prevented. Inhibitors ofEGF-R activation are proposed as therapy in hypersecretory airwaydiseases.

EXAMPLE 2 Role of Oxidative Stress in Production of Goblet Cells

In humans, prolonged cigarette smoking has been suggested to beassociated with progressive pathologic changes in peripheral airwaysincluding goblet cell hyperplasia. Likewise, experimental models ofcigarette smoking in animals have been shown to cause goblet cellhyperplasia in airways. However, the mechanism by which cigarette smokemay induce mucin synthesis is unknown. The following data demonstratethat proinflammatory cytokine-activated neutrophils and cigarette smokecause mucin MUC5AC synthesis in human bronchial epithelial cells vialigand-independentactivation of EGF-R. These results implicate recruitedneutrophils and cigarette smoke as regulators of epithelial celldifferentiation that may result in abnormal induction of mucin-producingcells in airways.

Methods

Isolation of Neutrophils. Human neutrophils were purified fromperipheral blood obtained from healthy human donors. Neutrophilisolation was performed by standard techniques of Ficoll-Hypaquegradient separation, dextran sedimentation, and hypotonic lysis oferythrocytes. Cells were routinely >95% viable by trypan blue dyeexclusion. To prevent endotoxin contamination, all solutions were passedthrough a 0.1 μm filter.

Cell Culture. NCI-H292 cells, a human pulmonary mucoepidermoid carcinomacell line, were grown in RPMI 1640 medium containing 10% fetal bovineserum, penicillin (100 U/ml), streptomycin (100 μg/ml) and Hepes (25 mM)at 37° C. in a humidified 5% CO₂ water-jacketed incubator. Either 6-wellculture plates or 8-chamber slides were used to culture the cells. Whenconfluent, cells were incubated for 1 h with neutrophils (10⁶ cells/ml)alone, TNFα alone (recombinant human TNFα, 20ng/ml, Genzyme, Cambridge,Mass.), IL-8 (recombinant human IL-8, 10⁻⁸ M, Genzyme) alone, fMLP (10⁻⁸M, Sigma, St. Louis, Mo.) alone, TNFα plus neutrophils, IL-8 plusneutrophils, fMLP plus neutrophils, hydrogen peroxide (H₂O₂, 200 μM),cigarette smoke solution or TGFα (recombinant human TGFα, 0.1-25 ng/ml,Calbiochem, San Diego, Calif.). The cells were then washed and incubatedwith fresh medium alone. Experiments were terminated at preselectedtimes (for mRNA, 6 h and 12 h; for protein, 24 h). As controls, cellswere incubated with medium alone for same time periods. In other studieswith neutrophils, TNFα was chosen as a stimulus because it had the mostpotent effect on MUC5AC synthesis. NCI-H292 cells were incubated for 1 hwith either neutrophils that had been incubated with TNFα (20 ng/ml) for1 h and then washed with sterile PBS to avoid contamination with thesupematant (e.g., molecules released from neutrophils), or the NCI-H292cells were incubated with supernatant only. In inhibition studies withEGF-R tyrosine kinase inhibitors, NCI-H292 cells were pretreated withBIBX1522 (10 μg/ml, generously provided by Boehringer Ingelheim Inc.,Ingelheim, Germany) or tyrphostin AG1478 (10 μM, Calbiochem) 30 minbefore adding a stimulus. The effects of a selective inhibitor ofplatelet-derived growth factor receptor tyrosine kinase (tyrphostinAG1295, 100 μM, Calbiochem), and a negative control for tyrphostins(tyrphostin A1, 100 μM, Calbiochem) were also examined. In inhibitionstudies with blocking antibodies to EGF-R ligands, the supernatants werepretreated with anti-TGFα antibody (Calbiochem) or anti-EGF antibody for30 min and then added to NCI-H292 cells. The role of oxygen freeradicals was examined using scavengers of oxygen free radical DMSO (1%,Sigma), 1,3-dimethyl-2-thiourea (DMTU, 50mM, Sigma), or superoxidedismutase (SOD, 300 U/ml, Sigma).

Preparation of Cigarette Smoke Solution. Research cigarettes (code2R1,produced for the University of Kentucky Tobacco and Health ResearchFoundation) were used in the study. Cigarette smoke solution wasprepared as previously described (Dusser et al. (1989) J. Clin. Invest.84:900-906). In brief, cigarette smoke was withdrawn into apolypropylene syringe (35 ml) at a rate of one puff/min (10 times) andbubbled slowly into 20 ml of RPMI1640 containing 50 mM Hepes buffer. Thesmoke solution was then titrated to pH 7.4 and used immediately afterpreparation.

Visualization of Mucous Glycoconjugates and MUC5AC Protein in NCI-H292Cells. At the end of experiments, the cells grown on 8-chamber slideswere fixed with 4% paraformaldehyde for 1 h and then either stained withAlcian blue/periodicacid-Schiff (PAS) to visualize mucousglycoconjugates, or used for immunocytochemistry of MUC5AC. Forimmunocytochemistry of MUC5AC, PBS containing O.05% Tween 20, 2% normalgoat serum and Levamisol (2 mM) was used as diluent for the antibody.Cells were incubated with mouse mAb to MUC5AC (clone 45 M1, 1:200, NeoMarkers, Fremont, Calif.) for 1 h at room temperature, and then washed 3times with PBS to remove excess primary antibody. Cells were thenincubated with biotinylated horse anti-mouse IgG (Vector LaboratoriesInc., Burlingame, Calif.) at 1:200 dilution for 1 h at room temperature.Bound antibody was visualized according to a standard protocol for theavidin-biotin-alkaline phosphatase complex method.

In Situ Hybridization for human MUC5AC gene. A 298 bp cDNA fragment ofhuman MUC5AC was inserted into TA cloning vector (Invitrogen, San Diego,Calif.). The preparation of RNA probes and in situ hybridization wereperformed as described above.

Immunoassay of MUC5AC Protein. MUC5AC protein was measured as describedabove. In brief, cell lysates were prepared with PBS at multipledilutions, and 50 μl of each sample was incubated withbicarbonate-carbonate buffer (50μl) at 40° C. in a 96-well plate(Maxisorp Nunc, Fisher Scientific, Santa Clara, Calif.), until dry.Plates were washed three times with PBS and blocked with 2% BSA(fraction V, Sigma) for 1 h at room temperature. Plates were againwashed three times with PBS and then incubated with 50μl of mousemonoclonal MUC5AC antibody (1:100) that was diluted with PBS containing0.05% Tween 20. After 1 h, the wells were washed three times with PBS,and 100 μl horseradish peroxidase-goat anti-mouse IgG conjugate(1:10,000, Sigma) was dispensed into each well. After 1 h, plates werewashed three times with PBS. Color reaction was developed with TMBperoxidase solution (Kirkegaard & Perry Laboratories, Gaithersburg, Md.)and stopped with 2N H₂SO₄. Absorbance was read at 450 nm.

Quantitative Analysis of TGFα Protein. TGFα protein was measured using acommercially available kit for ELISA (Sigma), following themanufacturer's instructions. Supernatant taken after incubation ofneutrophils plus TNFα (20 ng/ml) for 1 h was mixed with the lysis bufferPBS containing 1% Triton X-100, 1% sodium deoxycholate and severalprotease inhibitors, (Complete Mini, Boehringer Mannheim, Germany), andthen used to measure TGFα.

Immunoprecipitation for EGF-R Protein and Immunoblotting for TyrosinePhosphorylation. Cells were serum-starved for 24 h and then stimulatedwith TGFα, H₂O₂, or the supernatant of activated neutrophils for 15 min.After stimulation, cells were lysed and incubated for 30 min in anorbital shaker at 4° C. To remove insoluble material, cell lysates werecentrifuged at 14,000 rpm for 5 min at 4° C. Aliquots of supernatantscontaining equal amounts of protein were immunoprecipitated withanti-EGF receptor antibody (polyclonal, Ab4, Calbiochem) and 20 μl ofprotein A-agarose (Santa Cruz) for 2 h at 4° C. Precipitates were washedthree times with 0.5 ml of lysis buffer, suspended in SDS sample buffer,and boiled for 5 min. Proteins were separated by SDS-PAGE in 8.0%acrylamide gel. The resulting gel was equilibrated in the transferbuffer: 25 mM Tris-HCI, 192 mM glycine, 20% (vol/vol) methanol, pH 8.3.The proteins were then transferred electrophoretically to nitrocellulosemembranes (0.22 μm), blocked with 5% fat-free skimmed milk in PBScontaining 0.05% Tween 20 overnight and then incubated with monoclonalanti-phosphotyrosine antibody (1:100, Santa Cruz) for 1 h. Boundantibody was visualized according to a standard protocol for theavidin-biotin-alkaline phosphatase complex method (ABC kit, VectorLaboratories).

Statistics. All data are expressed as mean ± SEM. One-way analysis ofvariance was used to determine statistically significant differencesbetween groups. Scheffe's F test was used to correct for multiplecomparisons when statistical significances were identified in theanalysis of variance. A probability of less than 0.05 for the nullhypothesis was accepted as indicating a statistically significantdifference.

Results

Activated Neutrophils Cause Mucin MUC5AC Synthesis. When neutrophilsplus stimuli that activate neutrophils (IL-8, fMLP, TNFα) were incubatedwith NCI-H292 cells for 1 h, MUC5AC protein synthesis increasedsignificantly within 24 h, whereas non-stimulated neutrophils (10⁶/ml),IL-8 alone or fMLP alone showed no effect on MUC5AC synthesis;incubation with TNFα alone caused a small, insignificant increase inMUC5AC synthesis. When neutrophils were preincubated for 1 h with TNFα,and then the neutrophils and their supernatant were separated,subsequent incubation of the supernatant for 1 h with NCI-H292 cellsup-regulated MUC5AC gene expression within 12 h, and stimulated stainingwith both Alcian blue/PAS and with an antibody to MUC5AC protein within24 h; resting NCI-H292 cells showed little expression of MUC5AC gene andsmall, patchy staining of both Alcian blue/PAS and MUC5AC protein.MUC5AC protein synthesis induced by the supernatant increasedsignificantly from control; neutrophils separated from the supernatantafter incubation were without effect. It was concluded that activatedneutrophils rapidly secrete an active product, which causes MUC5ACsynthesis.

EGF-R Tyrosine Kinase Inhibitors Prevent MUC5AC Synthesis Induced bySupernatant of Activated Neutrophils. Because EGF-R ligands are known tocause MUC5AC synthesis in NCI-H292 cells via activation of EGF-Rtyrosine kinase, the role of EGF-R activation in MUC5AC synthesisinduced by the supematant of activated neutrophilswas examined.Pretreatment of NCI-H292 cells with selective EGF-R tyrosine kinaseinhibitors (BIBX1522, AG1478), prevented the MUC5AC protein synthesisthat was usually induced by the supernatant of activated neutrophils. Aselective platelet-derived growth factor receptor kinase inhibitor(AG1295) and a negative control for tyrphostins (A1) were withouteffect. These results implicate activation of EGF-R tyrosine kinase inMUC5AC synthesis induced by the supernatant of activated neutrophils.

Role of EGF-R Ligands Secreted in the Supernatant of ActivatedNeutrophils in MUC5AC Synthesis. To determine whether activation ofEGF-R tyrosine kinase is dependent on the EGF-R ligands (EGF and TGFα),we preincubated the supernatant of activated neutrophils withneutralizing antibodies to EGF-R ligands. Pretreatment of the supematantwith either anti-TGFα antibody or anti-EGF antibody did not inhibitMUC5AC synthesis induced by the supematantof activated neutrophils.Furthermore, TGFα was not detected in the supernatant. Thus, EGF-Rtyrosine phosphorylation caused by the supernatant of activatedneutrophils was induced by a mechanism independent of the EGF-R ligands,EGF and TGFα.

Cigarette Smoke and Oxygen Free Radicals Cause MUC5AC Synthesis.Cigarette smoke and the oxygen free radical, H₂O₂, up-regulated MUC5ACgene expression within 12 h, as did TGFα. Likewise, all stimuliincreased MUC5AC protein synthesis and mucous glycoconjugate productionwithin 24 h, effects that occurred in a dose-dependent fashion. Themaximum MUC5AC synthesis in response to H₂O₂ was significantly less thanthe response to TGFα. Pretreatment with AG1478 prevented the increase inMUC5AC protein synthesis induced by all stimuli, indicating that thestimuli cause mucin synthesis by the activation of EGF-R tyrosinekinase. MUC5AC synthesis by supernatant of activated neutrophils,cigarette smoke and H₂O₂ were significantly inhibited by pretreatmentwith free radical scavengers (DMSO and DMTU) and SOD, but MUC5AC proteinsynthesis by TGFα was unaffected by DMSO or SOD.

Induction of Tyrosine Phosphorylation of EGF-R by Supernatant ofActivated Neutrophils and by H₂O₂. The distribution of EGF-R protein wassimilar in serum-starved control and in all stimulated conditions(supernatant of activated neutrophils, cigarette smoke, H₂O₂ or TGFα).Total protein tyrosine phosphorylation occurred within 15 min afteradding supernatant of activated neutrophils, cigarette smoke, H₂O₂ orTGFα; the serum-starved control showed no effect. TGFα-induced totalprotein tyrosine phosphorylation was greater than the effect ofsupernatant of activated neutrophils, cigarette smoke or H₂0₂. Todetermine whether the EGF-R was phosphorylated, immunoprecipitation withanti-EGF-R antibody was performed: The supernatant of activatedneutrophils, the soluble products of cigarette smoke, and H₂O₂ allinduced EGF-R-specific tyrosine phosphorylation within 15 min, an effectthat was similar to that caused by TGFα. Pretreatment of NCI-H292 cellswith AG1478 inhibited EGF-R tyrosine phosphorylation by all stimuli.DMSO inhibited supernatant-, cigarette smoke-, and H₂O₂-induced EGF-Rtyrosine phosphorylation, but DMSO had no effect on TGFα-induced EGF-Rtyrosine phosphorylation.

The above results show that neutrophils cause mucin MUC5AC synthesis inNCI-H292 cells when they are activated with IL-8, fMLP, or TNFα.Moreover, the supernatant that was collected 1 h after the incubation ofneutrophils with TNFα caused MUC5AC synthesis, an effect that wasinhibited by selective EGF-R tyrosine kinase inhibitors. Inhibition ofEGF-R tyrosine kinase completely blocked MUC5AC synthesis caused by thesupernatant of activated neutrophils; a non-EGF-R tyrosine kinaseinhibitor, a selective platelet-derived growth factor receptor kinaseinhibitor (AG1295) and a negative control for tyrphostins (A1) werewithout effect, implicating EGF-R tyrosine phosphorylation as thesignaling pathway of MUC5AC synthesis induced by the supernatant ofactivated neutrophils.

To further analyze the mechanism by which supematants of activatedneutrophils induce EGF-R tyrosine phosphorylation, both ligand-dependentand ligand-independentEGF-R pathways were examined. First, we measuredTGFα in the supernatant of activated neutrophils and found that thesupernatant did not contain measurable amounts of TGFα. Previous reportsshowed that neutrophils only contained low concentrations (2.5 pg/10⁶cells) of TGFα. The effect of supernatant from activated neutrophils onMUC5AC synthesis was as potent as the effect of 1 ng of TGFα, which was400-fold higher than the amount of TGFα found in neutrophils. Second, weperformed blocking studies with neutralizing antibodies of EGF-Rligands: Pretreatment with neutralizing antibodies to EGF and TGFαfailed to inhibit MUC5AC synthesis caused by the supernatant ofactivated neutrophils. These results suggest that neutrophilsupernatant-induced MUC5AC synthesis was not due to the secretion ofEGF-R ligands (TGFα and EGF) by neutrophils. Next, we examined theligand-independent pathway: Because oxygen free radicals are known to bereleased by neutrophils during activation, and they are known to causetransactivation of EGF-R tyrosine kinase in various cells, wehypothesized that the release of oxygen free radicals by activatedneutrophils caused EGF-R tyrosine phosphorylation and resulting MUC5ACsynthesis in NCI-H292 cells. Scavengers of free radicals (DMSO and DMTU)and SOD inhibited MUC5AC synthesis by the supematant of activatedneutrophils. TNFα is reported to cause an oxidative burst in neutrophilsin suspension, with a maximum response within 1 h; the present resultsshow a similar time course.

In the present study, exogenous H₂O₂, a major product released fromneutrophils during oxidative burst, caused MUC5AC synthesis in NCI-H292cells. However, the maximum response to H₂O₂ in MUC5AC synthesis wasonly half of the response to TGFα. Asignificant finding in the presentstudy is the fact that cigarette smoke alone caused MUC5AC synthesis.This suggests that cigarette smoke could cause MUC5AC synthesis in vivoboth through direct stimulation and through indirect stimulation causedby recruitment of neutrophils. The exact molecules in cigarette smokecausing MUC5AC synthesis are still unclear. Cigarette smoke has beenshown to contain multiple products (eg., nicotine, tar, acrolein andoxidants). In our experiments, DMSO and SOD partially inhibited MUC5ACsynthesis induced by cigarette smoke. Thus, oxidant stress might be onemechanism producing this response. The fact that cigarette smoke-inducedMUC5AC synthesis was completely blocked by EGF-R tyrosine kinaseinhibitors indicates that EGF-R activation plays a principal role incigarette smoke-induced MUC5AC synthesis.

In airway diseases, neutrophilic airway inflammation is a commonfeature, and neutrophils are recruited and activated by cytokines and bycigarette smoke. The present studies show that recruited neutrophils andcigarette smoke also act as regulators of epithelial celldifferentiation that result in induction of mucin-producing cells inairways. Most importantly, inhibition of EGF-R activation will be usefulas therapy in hypersecretory airway diseases.

EXAMPLE 3 Wounding of Airway Epithelium Causes Goblet Cell Metaplasia

It was hypothesized that agarose plugs instilled into airways wouldlodge chronically in bronchi without obstructing them, and that residentplugs would cause inflammation, resulting in goblet cell metaplasia. Itis shown that agarose plugs induce marked local production of gobletcells, as shown by Alcian blue/PAS-positive staining and mucin MUC5ACgene expression, associated with local recruitment of inflammatorycells. The results implicate EGF-R activation in plug-induced gobletcell metaplasia.

Methods

Animals. The experimental animal protocol was approved by the Committeeon Animal Research of the University of California San Francisco.Specific pathogen-free, male F344 rats (230 to 250 g body weight;Simonsen Lab., Gilroy, Calif.) were used. The rats were housed inpathogen-free BioClean cages with environmentally controlled laminarflow hoods; animals had free access to sterile food and water.

Drugs. Drugs from the following sources were used: cyclophosphamide(Sigma, St. Louis, Mo.), methohexital sodium (Brevital, Jones MedicalIndustries, Inc., St. Louis, Mo.), pentobarbital sodium (Nembutal,Abbott Lab., North Chicago, Ill.); BIBX1522, a selective inhibitor ofEGF-R tyrosine kinase (generously provided by Boehringer Ingelheim,Inc., Ingelheim, Germany), was dissolved in the following solution: 2 mlpolyethylene glycol 400 (Sigma, St. Louis, Mo.), 1 ml 0.1 N HCI, and 3ml 2% mannitol solution in water (pH 7.0). NPC 15669 (an inhibitor ofleukocyte motility), was kindly provided by Scios Nova, Inc., MountainView, Calif.

Agarose plugs. Agarose plugs (0.7-0.8 mm diameter) were made with 4%agarose type II medium EEO (Sigma, St. Louis, Mo.) in sterilephosphate-buffered saline (PBS). To visualize the agarose plugs intissue, 3% suspension Monastral blue B (Sigma, St. Louis, Mo.) was addedafter melting the agarose at 50° C.

Protocol of experiments. We studied pathogen-free rats, because theynormally have few goblet cells in airways. The animals were anesthetizedwith methohexital sodium (Brevital, 25 mg/kg, i.p.). The trachea wasexposed aseptically with a midline cervical incision, and agarose plugswere instilled into a bronchus via a 20 gauge Angiocath (BectonDikinson, Sandy, Utah) connected to polyethylene tubing (PE 90, internaldiameter 0.86 mm and outer diameter 1.27 mm, Clay Adams, Parsippany,N.Y.) threaded into the incised trachea. The polyethylene tube was bentat a 300 angle to allow selective instillation into the right bronchus.After instillation, the incision was closed with a suture.

To evaluate the role of EGF-R on agarose plug-induced goblet cellmetaplasia, animals were treated with BIBX1522 (80 mg/kg, i.p.) 1 hbefore instillation of the agarose plugs and repeated daily (40 mg/kg,i.p., bid). Animals were euthanized 24, 48 or 72 h after instillation ofthe agarose plugs.

To evaluate the role of TNFα in agarose plug-induced goblet cellmetaplasia, animals were treated with a TNFα neutralizing antibody(Genzyme, Boston, Mass.). The first treatment (100 μl in 0.2 ml saline,i.p.) was given 1 h before the instillation of agarose plugs, and i.p.injections were repeated daily. In addition, TNFα neutralizing antibodywas infused (10 μl/h) via an osmotic minipump (Alzet 2ML1, Aiza Corp.,Palo Alto, Calif.) implanted subcutaneously.

To study the effect of neutrophils on agarose plug-induced goblet cellmetaplasia, rats were pretreated with cyclophosphamide (an inhibitor ofbone marrow leukocytes) or with a combination of cyclophosphamideplusNPC 15669. Cyclophosphamide(100 mg/kg, i.p.) was given 5d beforeinstillation of agarose plugs, and a second injection ofcyclophosphamide (50 mg/kg, i.p.) was given 1 d before instillation ofplugs. In studies with NPC 15669, the drug (10 mg/kg, i.p.) was injected1 h before instillation of agarose plugs, and then daily for 3 dthereafter.

All drugs (BIBX1522, TNFα neutralizing antibody, cyclophosphamide, andNPC 15669) were given i.p. 1 h before instillation of agarose plugs, anddoses were repeated daily for 3 d.

Tissue preparation. At various times after agarose plug instillation,rats were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), thesystemic circulation was perfused with 1% paraformaldehyde in diethylpyrocarbonate-treated PBS at a pressure of 120 mmHg. The right lung wasremoved, and the right caudal lobe was used for histology. For frozensections, tissues were removed and placed in 4% paraformaldehyde for 1 hand then replaced in 30% sucrose for cryoprotection overnight. Thetissues were embedded in O.C.T. compound (Sakura Finetek U.S.A., Inc.,Torrance, Calif.). For methacrylate sections, the tissues were placed in4% paraformaldehyde for 24 h and then dehydrated with gradedconcentrations of ethanol and embedded in methacrylate JB-4(Polysciences, Inc., Warrington, Pa.). Tissue sections (4 μm thick) werestained with Alcian blue/PAS and counterstained with hematoxylin.

Morphometric analysis of bronchial epithelium. The percentage of Alcianblue/PAS-stained area of mucous glycoconjugates in the epithelium wasdetermined using a semi-automatic image analysis system according topreviously published methods. The area of epithelium and Alcianblue/PAS-stained mucous conjugates within the epithelium was manuallycircumscribed and analyzed using the NIH Image program (developed at theU.S. National Institutes of health and available from the internet byananymous FTP or from a floppy disk from the National TechnicalInformation Service, Springfield, Va.; part number PB95-500195GEI). Thedata are expressed as the percentage of the total epithelial areaoccupied by Alcian blue/PAS stain. To evaluate mucus secretionsemi-quantitatively, the percentage of the length of epithelial surfaceoccupied by Alcian blue/PAS-positive staining was determined bycalculating the length that stained positively as a ratio to the totallength. The percentage of denuded epithelium was determined bycalculating the ratio of the length of denuded epithelium to the totalepithelial length.

Identification of cell types in methacrylate sections and cell analysis.The total number of epithelial cells was determined by countingepithelial cell nuclei over 2 mm of the basal lamina with an oilimmersion objective lens (×1000 magnification). The linear length of thebasal lamina under each analyzed region of epithelium was determined bytracing the contour of the digitalized image of the basal lamina. Theepithelial cells were identified as described previously. In brief,basal cells were identified as small flattened cells with a largenucleus, located just above the basal lamina but not reaching the airwaylumen. The cytoplasm stained darkly, and Alcian blue or PAS-positivegranules were not present. Ciliated cells were recognized by theirciliated borders, lightly stained cytoplasm, and large, round nucleus.Non-granulated secretory cells were columnar in shape and extended fromthe bronchial lumen to the basal lamina. After intrabronchialinstillation of agarose plugs, “developing” goblet cells (pre-gobletcells) were formed. These cells showed Alcian blue/PAS-positivestaining,the granules were small, and the cells were not packed with granules;they contained smaller mucous-stained areas (<⅓ height in epitheliumfrom basement membrane to luminal surface) or sparsely and lightlyAlcian blue/PAS-stained, small granules. Cells of indeterminate type aredefined as cell profiles lacking sufficient cytoplasmic characteristicsfor proper categorization.

Immunohistochemical localization of EGF-R. The presence of EGF-R wasdetermined by immunohistochemical localization, using a monoclonal mouseantibody to the EGF-R (Calbiochem, San Diego, Calif.). Previouslyprepared 4 Im frozen sections were post-fixed with 4% paraformaldehyde,treated with 0.3% H₂O₂/methanol. The tissues were incubated with EGF-Rantibody (1:250 dilution). Biotinylated horse anti-mouse IgG (1:200;Vector Lab., Burlingame, Calif.), followed by streptavidin-peroxidasecomplex (ABC kit, Vector Lab., Burlingame, Calif.) was used to visualizeantigen-antibody complexes stained with 3,3′- diaminobenzidinetetrahydrochloride (Sigma, St. Louis, Mo.). Negative control slides wereincubated with either the primary or secondary antibody omitted andreplaced with PBS.

In situ hybridization. [³⁵S]-labeled riboprobes were generated from aplasmid containing a 320 bp cDNA fragment of rat MUC5AC kindly providedby Dr. Carol Basbaum. Sections were hybridized with [³⁵S]-labeled RNAprobes (2,500-3,000 cpm/μl hybridization buffer) and washed understringent conditions, including treatment with RNase A. Afterautoradiography for 7-21 d, the photographic emulsion was developed, andthe slides were stained with hematoxylin.

Counting of neutrophils in airway epithelium. Evaluation of neutrophilinflux into bronchi was performed by staining neutrophils with3-3′-diaminobenzidine tetrahydrochloride, and the number of neutrophilswere counted in the airway lumen and in the epithelium; results wereexpressed as the number of stained cells per mm of basal lamina length.

Bronchoalveolar lavage(BAL). To assess differential cell counts in eachgroup of animals, lungs were lavaged five times with 3 ml aliquots ofsterile PBS, lavages were pooled, and the volume was measured. Cells inBAL were collected by spinning the lavage fluid at 1,000 rpm for 10 min.Ten microliters of a cell suspension was then counted with ahematocytometer to determine cell numbers in BAL fluid. Differentialcell counts were performed on cytospun preparations stained withDiff-Quik (American Scientific Products, McGaw Park, Ill.). Differentialcell counts were obtained by sampling at least 200 cells on eachcytospun slide.

Statistical analysis. Data are expressed as means ± SE. For statisticalanalysis, the two-way or one-way analysis of variance (ANOVA) followedby Student t test was used as appropriate. A probability of less than0.05 was considered a statistically significant difference.

Results

Effect on airway epithelial structure: Goblet cell metaplasia. Todetermine whether agarose plugs affect the structure of airwayepithelium, agarose plugs were instilled into the right bronchus in 5pathogen-free rats. In control animals, the bronchial epitheliumcontained few goblet cells. However, after local instillation of agaroseplugs, Alcian blue/PAS staining showed a time-dependent increase ingoblet cell area, which was detectable as early as 24 h and was greatest72 h after instillation. At 24 h, agarose plugs produced significantincreases in number of pre-goblet and goblet cells, and at 48 h moremature goblet cells were found (Table 1). At 72 h, agarose plugsincreased the number of goblet cells (P <0.01); the numbers of basal andciliated cells were not changed (P >0.05). The total number ofepithelial cells per mm basal lamina 72 h after instillation wasslightly but not significantly increased (P >0.05, Table 1); the heightof the epithelium (measured from basement membrane to luminal surface ofepithelium) was increased from 16.0±1.2 μm in control airways to38.1±9.1 μm at 72 h after instillation of plugs (n=5P<0.01).

In the airway lumen of control animals, there was no Alcian blue/PASstaining. However, adjacent to agarose plugs, positive staining was seenin the lumen, indicating that secretion of mucous glycoconjugates hadoccurred. In airways with agarose plugs, staining increasedtime-dependently. The percentage of the total length of epitheliumoccupied by Alcian blue/PAS-positive staining in airways adjacent toplugs increased from 0.1±0.1% in control animals to 4.7±1.4%, 13.3±0.7%,and to 19.1±0.7% at 24 h, 48 h, and 72 h (n=5). Furthermore, the agaroseplugs denuded the epithelium of the plugged bronchus by 13.5±2.3%,6.9±2.4%, and 5.1±1.5% of the total area at 24, 48, and 72 h,respectively (n=5).

Effect of agarose plugs on mucin gene expression. In control rats, therewas no detectable signal with the antisense probe of MUC5AC in bronchi(n=4 per group). In bronchi where agarose plugs were instilled, therewas a signal for MUC5AC that increased time-dependently from 24 to 72 h(n=4). MUC5AC gene expression was found preferentially in cells thatstained positively with Alcian blue/PAS. No signals were detected inother cell types (e.g., smooth muscle, connective tissue). Sectionsexamined with the MUC5AC sense probe showed no expression.

Effect of agarose plugs on EGF-R expression in airway epithelium. Incontrol animals, immunostaining with an antibody to EGF-R showed sparsestaining in epithelium. However, after instillation of agarose plugs,epithelium adjacent to agarose plugs showed EGF-R-positive staining incells that stained positively with Alcian blue/PAS. The staining patternfor EGF-R paralleled the staining for MUC5AC and AB/PAS. Pre-goblet,goblet, and non-granulated secretory cells were immunopositive forEGF-R. Ciliated cells showed no immunoreactivity. In airways notobstructed by agarose plugs, the epithelium showed little staining forEGF-R and appeared similar to staining in control animals.

Effect of EGF-R tyrosine kinase inhibitor on goblet cell metaplasia andon mucin gene expression. In the present studies, instillation ofagarose plugs resulted in the expression of EGF-R in the cells thatproduce mucins. EGF-R is a member of the class of tyrosine kinasereceptors. Thus, when the EGF-R ligands (EGF or TGFα) bind to EGF-R, aspecific EGF-R tyrosine kinase is activated. Therefore, to test thehypothesis that EGF-R activation induces expression of MUC5AC gene andof mucous glycoconjugates after instillation of agarose plugs, an EGF-Rtyrosine kinase inhibitor (BIBX1522) was injected intraperitoneally inrats. BIBX1522 markedly inhibited agarose plug-induced Alcianblue/PAS-stained area of epithelium at 24, 48 and 72 h. It alsocompletely inhibited the expression of MUC5AC gene at 72 h after pluginstillation.

Effect of TNFα neutralizing antibody on goblet cell metaplasia and onEGF-R protein expression. We hypothesized that TNFα is released duringthe inflammation caused by agarose plugs. Therefore, we examined theeffect of pretreatment of rats with a TNFα neutralizing antibody onagarose plug-induced goblet cell metaplasia: In animals pretreated withthe TNFα neutralizing antibody (n=5), agarose plugs no longer stimulatedEGF-R protein expression or the production of Alcian blue/PAS-positivelystained (goblet) cells.

Inflammatory cell recruitment by agarose plugs. It was noted thatagarose plugs cause epithelial damage and inflammatory cellinfiltration. Various inflammatory cells can produce both TNFα and EGF-Rligands. Both EGF-R and its ligands are involved in the EGF-R cascadethat leads to goblet cell metaplasia. We evaluated the roles ofleukocytes and macrophages in agarose plug-induced effects in two ways.First, we examined cells in bronchoalveolar lavage: In control rats,macrophages were the predominant cells recovered (n=5; FIG. 4, Control).After instillation of agarose plugs, the number of macrophages increased(P<0.05), and significant numbers of neutrophils (P<0.01) appeared inthe lavage fluid. The number of lymphocytes was unchanged.

Infiltrating cells were also evaluated in tissue sections: airwayswithout agarose plugs contained few neutrophils, but airways containingplugs showed presence of neutrophils, both in the epithelium and in thelumen. The number of neutrophils in the airway lumen was 0.2±0.2,42.4±7.1, 40.7±7.7, and 20.1±7.2/mm of basal lamina in control airwaysand at 24, 48, and 72 h after instillation of plugs, respectively(P<0.05, n=5). In addition, the number of neutrophils in airwayepithelium was 1.3±0.4, 15.6±2.6, 14.9±1.4, and 14.8±2.6/mm of basallamina in control and at 24, 48, and 72 h after in of plugs,respectively (P<0.01, n=5).

Effect of cyclophosphamide on neutrophil recruitment, goblet cellmetaplasia, and EGF-R protein expression. In cyclophosphamide-treatedrats, blood neutrophils were depleted (neutrophil count in venous bloodafter cydophosphamide, 1.8±0.5%, n=5), and plug-induced neutrophilrecruitment in BAL was inhibited. The number of neutrophils in theairway lumen (2.6±0.3/mm of basal lamina) and in the epithelium(0.8±0.2/mm) also decreased significantly at 24 h. Cyclophosphamide alsoinhibited agarose plug-induced goblet cell metaplasia and the expressionof EGF-R protein. When the leumedin, NPC 15669 was added tocyclophosphamide, the inhibition of agarose plug-induced goblet cellmetaplasia was similar to the effect of cyclophosphamidealone. Theseresults implicate neutrophils in plug-induced goblet cell metaplasia.

Discussion

In the present study, we examined the effect of instillation of agaroseplugs on goblet cell metaplasia in airways of pathogen-free rats, whichhave very few goblet cells in the control state. Epithelial cells inbronchi in control animals and bronchi without agarose plugs (controllungs) stained uniformly negatively with Alcian blue/PAS. Instillationof agarose plugs resulted in a profound, time-dependent increase ingoblet cell area of bronchial epithelium adjacent to the instilledplugs, which was detectable within 24 h and was greatest approximately72 h after instillation. Airways adjacent to plugged airways alsostained positively with Alcian blue/PAS. The total cell number and thenumber of basal and ciliated cells did not change, but the number ofgoblet cells increased, and the number of non-granulated secretory cellsdecreased time-dependently after agarose plug instillation (Table 2).These results suggest that the goblet cell metaplasia was the result ofconversion of non-granulated secretory cells to goblet cells.

TABLE 2 Effect of agarose plugs on the distribution of bronchialepithelial cells in pathogen-free rats*. Cell Type Control 24 h 48 h 72h Goblet 0.0 ± 0.0 13.1 ± 5.6  25.7 ± 15.0 51.5 ± 9.0 Pre-Goblet 0.0 ±0.0 32.8 ± 2.9  25.7 ± 15.0 51.5 ± 9.0 Secretory 43.5 ± 3.0  24.4 ± 3.318.4 ± 3.7  8.9 ± 2.3 Ciliated 98.5 ± 4.0  83.8 ± 7.9 81.6 ± 5.0 84.0 ±3.9 Basal 18.4 ± 5.7  10.6 ± 0.8 11.6 ± 1.7 11.0 ± 2.3 Indeterminate^(†)1.3 ± 0.5  1.4 ± 0.8  1.1 ± 0.1  0.6 ± 0.4 Total 161 ± 7.2  166.1 ± 6.1 175.5 ± 6.2  180.9 ± 7.5  Cells were analyzed as described in Methods; n= 5 in each group. Characterization was aided by Alcian blue/PASstaining (which stains mucous glycoconjugates). Control airwayscontained few pre-goblet and goblet cells. After instillation of agaroseplugs, there was a time-dependent (24, 48, 72 h) increase in the numberof pre-goblet and goblet cells, and a decrease in the number ofnon-granulated secretory cells compared to control animals. *Data aremeans ± SE, number of cells/mm basal lamina. ^(‡)P < 0.05 compared tocontrol. ^(§)P < 0.01 compared to control. Cells lack sufficientcytoplasmic characteristics for categorization.

Rat airway goblet cells are reported to express the MUC5AC gene. In thepresent studies, control bronchi did not express MUC5AC gene, butairways obstructed by plugs or adjacent to the plugs, which stainedpositively with Alcian blue/PAS, expressed the MUC5AC gene, suggestingthat MUC5AC gene is involved in agarose plug-induced mucus production.These results indicate that agarose plugs induce the expression of mucingenes and the production of mucous glycoconjugates in selected cells inrat airways.

The mechanism of goblet cell metaplasia induced by agarose plugs wasexamined. EGF-R are not normally expressed in airway epithelium ofpathogen-free rats but is induced by TNFα. In the presence of EGF-R inepithelium, instillation of EGF-R ligands (EGF or TGFα) results in anincrease in mucin gene and protein expression. A selective inhibitor ofEGF-R tyrosine kinase (BIBX1522) completely inhibits these responses,implicating EGF-R signaling in goblet cell metaplasia. The effect ofBIBX1522 on agarose plug-induced goblet cell metaplasia was determined:BIBX 1522 inhibited agarose plug-induced production of mucousglycoconjugates and MUC5AC gene expression. These results implicateanEGF-R cascade in agarose plug-induced goblet cell metaplasia.

The mechanisms by which the EGF-R cascade causes goblet cell metaplasiawith agarose plugs were studied. First, we studied the expression ofEGF-R protein in the bronchial epithelium. Control airways staineduniformly negatively for EGF-R, but airways containing agarose plugsshowed selective, time-dependent positive staining for EGF-R. Positivelystained cells included non-granulated secretory, pre-goblet, and gobletcells. Thus, agarose plugs induced EGF-R protein expression. Rats thatwere pre-treated with a neutralizing antibody to TNFα did not developagarose plug-induced goblet cell metaplasia, implicating TNFα in agaroseplug-induced EGF-R expression.

Cyclophosphamide, a drug that selectively depresses leukocyteproduction, prevented neutrophil recruitment into airway lavage fluidand into the airway epithelium following the instillation of agaroseplugs and also prevented agarose plug-induced goblet cell metaplasia.Macrophages were also increased after the introduction of agarose plugs,but cyclophosphamide did not inhibit macrophage recruitment. Theseresults implicate neutrophils in agarose plug-induced goblet cellmetaplasia. The fact that cydophosphamide also decreased EGF-R proteinexpression after agarose plugs suggests that neutrophils contribute, atleast in part, to the EGF-R expression in this inflammatory condition.

Neutrophils are also capable of producing the EGF-R ligands, EGF andTGFα. In addition, epithelial cells are sources of EGF-R ligands, andthere was striking denudation of epithelium adjacent to the agaroseplugs. Thus, the epithelium could be an important potential source ofboth TNFα and EGF-R ligands.

It is reasonably assumed that the effective stimulus of the agarose plugis related to movement of the plugs during breathing, with subsequentepithelial abrasion. Mechanical injury to airway epithelium has beenreported to cause hypersecretion. These prior studies lend credence tothe hypothesis that mechanical trauma to the airway epithelium leads tohypersecretion. Orotracheal intubation is reported to result in abundantmucus secretion in horses. Chronic intubation in patients could causemucous hypersecretion and could be responsible for mucous plugging.Inhibitors of EGF-R tyrosine kinase could serve to prevent mucoushypersecretion after tracheal intubation.

Epithelial damage is a common finding in studies of patients even withmild asthma, and the damage is increasingly related to worsening ofclinical symptoms. Epithelial damage produced by the allergic responsemay induce EGF-R activation, which results in abnormal goblet cellproduction. The data presented above implicate EGF-R activation in adifferent response, specifically involving goblet cell metaplasia.Mechanical epithelial damage and epithelial injury in asthma may involvea similar (EGF-R) cascade, resulting in abnormal growth of epithelialsecretory cells. This provides a mechanism for the hypersecretion thatoccurs in fatal cases of acute asthma.

EXAMPLE 4 Recranulation of Goblet Cell by EGF- Receptors

Degranulation of goblet cells in rat nasal respiratory epithelium wasinduced by intranasal inhalation of fMLP. Significant degranulation wasinduced in the nasal septal epithelium 4 h after intranasal inhalationof fMLP (10⁻⁷M). Goblet cell regranulation occurred by 48 h afterinhalation. In the control state, MUC5AC protein was expressed in thegoblet cells, but EGF-R protein was not expressed. Both EGF-R and MUC5ACmucin gene and protein were absent in control epithelium but wereexpressed significantly 48 h after inhalation. Pretreatment with anEGF-R tyrosine kinase inhibitor, BIBX1522, inhibited mucin MUC5AC geneand protein expression following fMLP-induced goblet cell degranulation.These results indicate that EGF-R expression and activation are involvedin regranulation of goblet cell in rat nasal epithelium.

Methods

Animals. The experimental animal protocol was approved by the Committeeon Animal Research of the University of California San Francisco.Specific pathogen-free male F344 rats (200 to 230 g body weight;Simonsen Lab, Gilroy, Calif.) were used. The animals were housed inpathogen-free BioClean cages with environmentally controlled laminarflow hoods; animals had free access to sterile food and water.

Nasal Tissue Preparation. At various times after inhalation, rats wereanesthetized with pentobarbital sodium (65 mg/kg, i.p.). The heart ofthe animal was exposed, a blunt-ended needle was inserted from the apexof the left ventricle into the ascending aorta, and the systemiccirculation was perfused with 1% paraformaldehyde. An incision in theright atrium provided an outlet for the fixative. The eyes, lower jaws,skin, and musculature were removed, and the head was immersed in a largevolume of the same fixative for 24 h. After fixation, the head wasdecalcified with Surgipath (Decalcifier II, Surgical Medical Industries,Inc., Richmond, Ill.) for 4-5 days and rinsed in phosphate-bufferedsaline. The nasal cavity was sectioned transversely at the level of theincisive papilla of the nasal palate. The frontal tissue block wasembedded in glycol methacrylate (JB 4 Plus, Polysciences, Inc.,Warrington, Pa.), or in OCT compound (Sakura Finetek, U.S.A., Inc.,Torrance, Calif.) for frozen sections. Five μm-thick sections were cutfrom the anterior surface of glycol methacrylate-embedded blocks andstained with either Alcian blue (pH 2.5)/periodic acid-Schiff (AB/PAS)to demonstrate acid and neutral glycoconjugates, or 3,3′-diaminobenzidine (Sigma chemical, St. Louis, Mo.) to visualizeleukocytes that had migrated into the epithelium. Five μm-thick sectionswere cut from the anterior surfaces of frozen-embedded blocks andstained with AB/PAS or used for immunostaining of EGF-R and MUC5AC.Counting of Neutrophils in Nasal Epithelium. Neutrophils were counted inhigh power fields of the epithelial layer stained with3,3′-diaminobenzidineat magnificationx×400. The number of neutrophilswithin the nasal sepal epithelium (from the basement membrane to cellapices) was determined by counting the number of nuclear profiles perunit of basal lamina length.

Quantification of Goblet Cell Degranulation and Regranulation. To assessgoblet cell degranulation and regranulation, we measured the volumedensity of Alcian blue/PAS-stained mucosubstances on the mucosal surfaceepithelium using a semiautomatic imaging system according to apreviously published method. We examined the stained slides with anAxioplan microscope (Zeiss, Inc.), which was connected to a video cameracontrol unit (DXC755OMD; Sony Corp. of America, Park Ridge, N.J.).Images of the nasal epithelium were recorded in high power fields with aphase contrast lens at×400, using an IMAXX Video System (PDI, Redmond,Wash). The intracellular mucin in superficial epithelial secretory cellsappears as oval-shaped, purple granules of varying sizes. We measuredAlcian blue/PAS-positive-stained area and total epithelial area, and weexpressed the data as the percentage of Alcian blue/PAS area to totalarea. The analysis was performed on a Macintosh 9500/120 computer (AppleComputer, Inc., Cupertino, Calif.), using the public domain NIH Imageprogram.

Immunolocalization of EGF-R and MUC5AC protein. Frozen sections from theparaformaldehyde-fixed nasal tissues were treated with 3% H₂O₂/methanolto block endogenous peroxide and were incubated with a mouse monoclonalantibody to EGF-R (Calbiochem, San Diego, Calif.), or MUC5AC (NeoMarkersInc., Fremont, Calif.) for 1 h at a dilution of 1:100. ImmunoreactiveEGF-R or MUC5AC was visualized with the Vectastain Elite ABC kit (VectorLab., Inc., Burlingame, Calif.) using 3,3-diaminobenzidinetetrahydrochloride as a chromogen. Controls included the substitution ofprimary or secondary antibody with PBS.

Methods. We studied pathogen-free rats, which normally have many gobletcells in the nasal septal epithelium. To determine the effect ofaerosolized fMLP on goblet cell degranulation and on neutrophilmigration into nasal mucosa epithelium, the animals were anesthetizedwith sodium pentobarbital (65 mg/kg, i.p.), and they receivedN-formyl-methionyl-leucyl-phenylalanine (FMLP; 10⁻⁵ M, Sigma, St. Louis,Mo.) in pyrogen-free saline intranasally by aerosol for 5 min. Aerosolexposure was accomplished by ventilating the animals with an ultrasonicnebulizer (PulmoSonic, DeVilbiss Co., Somerset, Pa.) that generated anaerosol mist at rate of 0.3 ml/min. Similarly, control animals weregiven saline aerosol alone intranasally.

To study regranulation of nasal goblet cells after inhalation of fMLPaerosol, the rats were euthanized 48 h after intranasal delivery offMLP.

To evaluate the effect of EGF-R tyrosine kinase activation on gobletcell re-granulation, animals were pretreated intraperitoneallywith anEGF-R tyrosine kinase inhibitor (BIBX1522, 15 mg/kg, generously providedby Boehringer Ingelheim Inc., Ingelheim, Germany) 30 min before theinhalation of fMLP and repeated twice a day.

Statistics. All data are expressed as mean ± SEM. The one-way ANOVA orstudent t test was used for each experimental group. A probability ofless than 0.05was considered a statistically significant difference.

Results

Effect of goblet cell degranulation by fMLP on nasal epfthelialstructure. In the control state, the nasal septal epithelium contained asignificant area of AB/PAS-stained goblet cells, but the luminal surfacewas unstained. Immunostaining for mucin MUC5AC protein corresponded tothe area of AB/PAS staining, but in situ hybridization showed little orno MUC5AC gene expression. Immunohistochemical staining for EGF-Rprotein was negative. These results indicate that control rat nasalepithelium contains intact goblet cells containing MUC5AC protein in theabsence of expression of the mucin gene. The absence of luminal stainingsuggests that degranulation (secretion) of mucins was not present.

It was hypothesized that non-stimulated rat nasal epithelium contains“stable”, non-degranulating goblet cells containing mucin proteins.Neutrophil chemoattractants (e.g., fMLP) have been shown to recruitneutrophils into the airway epithelium, where they cause GCdegranulation via an elastase- dependent process. To examine the effectof GC degranulation, an aerosol of the neutrophil chemoattract, fMLP(10⁻⁷M) was delivered intranasally for 5 min. In rats euthanized 4 hafter fMLP, the AB/PAS-stained area and the area of MUC5AC-positiveimmunostaining were markedly decreased and neutrophil recruitment intothe nasal epithelium occurred. AB/PAS staining was prominent on thenasal airway luminal surface, confirming that GC degranulation had takenplace. At 4 h after fMLP, however, MUC5AC gene expression was unchanged.

In rats euthanized 48 h after fMLP, immunohistochemical staining with anEGF-R antibody stained positively for EGF-R in pre-goblet and gobletcells the area of AB/PAS- and MUC5AC-immunopositive staining returned tothe level present in the control state, indicating that regranulation ofthe nasal GC had occurred. At this time, neutrophil recruitment was nolonger present; MUC5AC gene expression was visible in the area occupiedby the GC, indicating that the GC regranulation was associated withincreased mucin gene expression.

Role of EGF-R tyrosine kinase phosphorylation in goblet cellregranulation. Previous studies in rats reported that activation ofEGF-receptors (EGF-R) leads to mucin gene and protein expression. Totest the hypothesis that EGF-R activation plays a role in rat nasal GCregranulation after fMLP, rats were pretreated (n=5) with the selectiveEGF-R tyrosine kinase inhibitor, BIBXL 522; aerosolization of fMLPcaused neutrophil recruitment into the nasal epithelium and GCdegranulation, but 48 h later the areas of AB/PAS staining andMUC5AC-immunopositive staining remained decreased. These resultsimplicate EGF-R activation in nasal GC mucin synthesis followingdegranulation by fMLP.

In the present study, we examined the regulation of mucin production inrat nasal epithelium. Control epithelium contained a significant numberof goblet cells, and MUC5AC protein was present in these cells. However,MUC5AC gene expression was absent. MUC5AC mucin expression is reportedto occur in other airway epithelial cells via the expression of EGF-Rand their activation. In control rat nasal epithelial cells, we found noEGF-R gene or protein expression. There was no luminal staining forAB/PAS or MUC5AC protein, suggesting that significant goblet celldegranulation (secretion) was not taking place. EGF-R might bedown-regulated in “stable” goblet cells, preventing further mucinsynthesis. Therefore, we “challenged” the nasal goblet cells by inducinggoblet cell degranulation, and we examined the subsequent changes inairway epithelial structure.

Neutrophil chemoattractants cause neutrophil-dependent goblet celldegranulation in guinea pig and human airways mediated by neutrophilelastase, involving close contact between neutrophils and goblet cells.To induce the degranulation of normally present goblet cells in thenasal septum, the chemoattractant, fMLP was inhaled intranasally. fMLPrecruited neutrophils in the nasal epithelium, followed by degranulationof the goblet cells; Alcian blue/PAS-stained area decreased markedly.

Next, we examined the subsequent events in the nasal epithelium afterfMLP-induced GC degranulation. At 4h after fMLP, when maximumdegranulation of nasal GC occurred, EGF-R and MUC5AC expression remainedabsent. However, 48h after fMLP, EGF-R was strongly expressed inpregoblet and goblet cells. MUC5AC gene expression was now stronglyexpressed in the epithelium, and these events were associated withregranulation of the goblet cells (increased AB/PAS and MUC5ACstaining). In fact, 48h after fMLP, regranulation had occurred to thepoint that the goblet cell area was similar to the control state. Thesefindings suggest that GC degranulation leads to expression andactivation of EGF-R, thus inducing mucin MUC5AC expression.

To examine the role of EGF-R tyrosine kinase activation in GCregranulation, we pretreated animals with a selective EGF-R tyrosinekinase inhibitor, BIBX1522. In animals pretreated with BIBX1522, fMLPstill caused GC degranulation. However, pretreatment with BIBX1 522prevented the regranulation of the GC and their expression of MUC5ACprotein. These results implicate EGF-R activation in the re-growth ofmucins after GC degranulation. In pathogen-free rats goblet cells are“inactive” (ie, not degranulating) and EGF-R are down-regulated. Wheninflammation (eg, stimulation of neutrophil infiltration) causes GCdegranulation and mucin secretion, up-regulation and activation of EGF-Rre-supplies the airway epithelium with mucins. The present findingssuggest that selective EGF-R tyrosine kinase inhibitors may be useful inpreventing hypersecretion in nasal disease.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An in vitro method for screening candidate agentsfor therapeutic potential in treating mucus hypersecretion in an airway,comprising: (i) contacting an in vitro model of goblet celldifferentiation with an epidermal growth factor receptor (EGFR) ligand;(ii) subsequently contacting the in vitro model with the candidateagent; and (iii) assessing goblet cell differentiation; wherein adecrease in goblet cell differentiation is indicative of the candidateagent's therapeutic potential.
 2. The in vitro method of claim 1,wherein the in vitro model comprises lung epithelial cells.
 3. An invivo method of screening candidate agents for therapeutic potential intreating mucus hypersecretion in an airway, the method comprising: (i)providing an in vivo mammalian model of hypersecretory pulmonary diseaseby inducing epidermal growth factor receptor (EGF-R) expression inairway epithelial cells of the mammalian model; (ii) stimulating theexpressed EGF-R in the airway epithelial cells of the mammalian modelcomprising administering an EGF-R ligand to said airway epithelialcells, whereby mucin producing goblet cells are produced; (iii)administering a candidate agent to said mammalian model; and (iv)assessing goblet cell differentiation or mucus secretion in the airwayof the mammalian model; wherein an inhibition of goblet celldifferentiation or mucus secretion is indicative of the candidateagent's therapeutic potential.
 4. The in vivo method of claim 3, whereinthe mammal used in the in vivo model is selected from the groupconsisting of mouse, rat, rabbit or guinea pig.
 5. The in vivo method ofclaim 3, wherein the in vivo model is an asthmatic model.